Cross-linked star-shaped self-assembled polypeptides and its use as carriers in biomedical applications

ABSTRACT

The invention relates to 3-arms star-shaped polypeptides derivatives which are able to self-assemble to form bioresponsive nanometric globular structures with controllable size and shape. These multivalent constructs also present the ability of disassemble under specific physiological conditions and of linking to at least one active agent so that they can be used as carries in biomedical applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application is a national phase application ofInternational Application No. PCT/EP2016/067554, filed Jul. 22, 2016,which claims priority to European Application No. 15382422.2, filed Aug.7, 2015, the disclosures of which are incorporated herein by reference.

The invention relates to 3-arm star-shaped polypeptides derivativeswhich are able to self-assemble to form bioresponsive nanometricglobular structures with controllable size and shape. These multivalentconstructs also present the ability of disassemble under specificphysiological conditions and of linking to at least one active agent sothat they can be used as carries in biomedical applications.

BACKGROUND ART

There has been a considerable effort devoted to the development of newand more versatile polymeric architectures with specific and predictableproperties to be used as targeted drug delivery systems. Such desirablefeatures in these materials include: adjustable molecular weights(higher molecular weight (MW), to enhance passive targeting by theEnhanced Permeability and Retention (EPR) effect), predictable structureand conformation in solution, lower heterogeneity, and greaterpossibility for multivalency. Nevertheless, the design and synthesis ofnew polymeric constructs of relevant MW, together with theirphysicochemical characterization, conformational studies, and especiallytheir potential for biological applications still remain to be fullyexploited in this area. To this aim, polypeptide-based architectures canbe considered suitable aspirants.

Star polypeptides are branched polymers, which consist of various linearchains linked to a central core. There are two main synthetic strategiesdescribed: the core-first approach (or multifunctional initiators ordivergent approach) and the arm-first approach (or the use ofmultifunctional linking agents, or convergent approach). Variouspolypeptide-based star polymers have been synthesized over the years.For example, Klok et al. (Journal of Polymer Science Part A: PolymerChemistry 2001, 39, (10), 1572-1583) used perylene derivatives with fourprimary amine groups as initiators to lead 4-armpoly(gamma-benzyl-L-glutamate) (PBLG) andpoly(epsilon-benzyloxy-carbonyl-L-lysine) (PZLL) and Inoue et al.(Macromolecular Bioscience 2003, 3, (1), 26-33) used hexafunctionalinitiators for the synthesis of 6-arm PBLG star polymers both takingprofit of the Ring Opening Polymerization (ROP) of N-Carboxyanhydrides(NCAs) techniques. Other examples are provided from the work of Aliferiset al. (Macromolecular Symposia 2006, 240, (1), 12-17) who used2-(aminomethyl)-2-methyl-1,3-propanediamine as a trifunctional initiatorfor the synthesis ofPoly(epsilon-carbobenzoxy-L-lysine-block-γ-benzyl-L-glutamate),P(BLL-b-BLG)₃ 3-arm star-block co-polypeptides; or the studies ofKaratzas et al. (Reactive and Functional Polymers 2009, 69, (7),435-440) in the synthesis of 4-arm poly(ethyleneoxide)-block-poly(y-benzyl-L-glutamate) (PEO-b-PBLG) hybrid star blockco-polymers using 4-arm PEO stars end-functionalized with primary aminesas initiators for the polymerization of gamma-Benzyl-L-Glutamate NCA(BLG-NCA) among others. Besides these two widely used approaches, alatest classification takes into account a new synthetic strategy. Thisapproach consists on the reaction of living macroinitiators (MI) (alsonamed macromonomers) with multifunctional molecules acting ascross-linkers giving rise to star-shaped architectures known as corecross-linked star (CCS) polymers (Chen et al. Macromolecular RapidCommunications, 2013, 34, 1507)

One of the most appealing properties, apart from their rheologicalcharacteristics and thermoplastic character, is their self-assemblybehavior that can be promoted in solution by the presence of functionalmoieties along the chain arms (in the case of homopolymers) or by usingselective solvents (in the case of star-blocks or miktoarm stars).Micellar structural parameters such as critical micellar concentration(CMC), aggregation number, core and shell dimensions, overall micelleconcentration as well as thermodynamics and kinetics of micellization ofcomplex structures, such as star-block copolymers and miktoarm stars,have been poorly investigated if compared to linear analogues. Ingeneral basis, star structures have higher CMC values and consequently,lower aggregation numbers than their linear block copolymerscounterparts.

Overall, it is well-known that macromolecular architecture is a keyparameter for the tuning of micellar behavior and properties, and thus,it must be well-considered for the design of new materials and theirpotential biological applications, in particular as drug deliverysystems.

Moreover, despite the growing interest in the development of hybrid andpeptide-based star polymers as prospective advanced materials forbiological applications, only recently, they have been explored as drugdelivery systems. For instance, Sulistio et al. (Chem. Commun, 2011, 47,1151-1153), synthesized highly functionalized water soluble corecross-linked star (CCS) polymers having degradable cores synthesizedentirely from amino acid building blocks which are capable ofencapsulating water-insoluble drugs. These types of stars were able toentrap hydrophobic drugs, such as the anti-cancer drug pirarubicin,through physical interactions with pyrene moieties of the core.Moreover, due to the presence of disulfide bonds at the core, the starscould also be cleaved by reducing agents such as dithiothreitol,yielding redox-sensitive polymers.

DESCRIPTION OF THE INVENTION

The present invention relates to a family of 3-arms star shapedpolypeptide derivatives consisting of a 1,3,5-Benzenetricarboxamiderelated central core employed as the initiator for the ring openingpolymerization of N-carboxyanhydride monomers and 3 polypeptide backbonearms. These systems undergo a self-assembly process yielding structuresin the nanometer range (4-300 nm in radius). Post-polymerizationmodifications of the polypeptide residues leads to the introduction ofcross-linking groups convenient for the stabilization of the resultingself-assembled nanostructures which can be further functionalized withthe introduction of one or more active agents for multiple applicationsin biomedicine.

A first aspect of the present invention is related to a compound offormula (I) below, comprising homo-polypeptides or random or blockco-polypeptides:

or its salts, solvates or isomers, wherein:m, m′, m″, n, n′, n″, o, o′ and o″ are integers independently selectedfrom 0 to 500, wherein at least one of them is ≥1;R₆ to R₈, R_(6′) to R_(8′) and R_(6″) to R_(8″) are independentlyselected from H and methyl;I₁ to I₃ are independently selected from the group consisting of H;halogen; Deuterium; and (C₁-C₂₀)-alkyl;each R₂ to R₄, R_(2′) to R_(4′), and R_(2″) to R_(4″) represents theside residues of amino acids in the polypeptidic backbone obtained bymeans of the ROP of NCAs, being either block or random copolypeptides,and each of them is independently selected from the group consisting of:

X₁ and X₂ are independently selected from the group consisting of H; N;NH_(2′); and Z;X₃ and X₄ are independently selected from the group consisting of H; andZ;y and y′ are integers between 0 and 3; and y+y′=2 or 3;y″ and y′″ are integers between 0 and 2; and y+y′=1 or 2Z is selected from the group consisting of H; metallic counterion;inorganic counterion; and an amino acid protecting group. In the presentinvention, the expression “amino acid protecting group” refers to anychemical functional group which can be introduced to the amino acid ofthe molecule by chemical modification to obtain chemoselectivity in asubsequent chemical reaction. It plays an important role in multisteporganic synthesis. The person skilled in the art knows the amino acidprotecting groups, some of them are detailed in Chem Rev 2009, 109,2455-2504, incorporated here by reference.R₁, R_(1′) and R_(1″) are radicals independently selected from the groupconsisting of:

A₁, A₂, A₃ and A₄ denote the side residues of amino acids, and areindependently selected from the group as defined for R₂ to R₄, R_(2′) toR_(4′), and R_(2″) to R_(4″);L₁ is a radical selected from the group consisting of (C₁-C₅₀₀)-alkyl,wherein one or more H is optionally substituted by: (1)(C₃-C₃₀)-cycloalkyl, (2) a C-radical derived from a ring system with 1-6rings, each ring being independently saturated, partially unsaturated oraromatic, the rings being isolated or fused and having 3-20 members eachmember independently selected from the group consisting of C, CH, CH₂,CO, N and NH, (3) OH, (4) NR_(a)R_(b), (5) ONR_(c)R_(d), (6) CN, (7)halide, (8) SH₂, (9) SR_(e)R_(f), (10) N(H)NH₂, (11) R_(g)COR_(h), (12)COOR_(i), (13) CON(R_(j))(R_(k)), (14) R_(l)N(R_(m))CON(R_(n))₂, (15)(C₁-C₃₀)-alkene, (16) (C₁-C₃₀)-alkyne, (17) N₃, (18)R_(o)CH(OR_(p))(OR_(q)), (19) R_(r)CH(SR_(s))(SR_(t)), (20)R_(u)Boron(OR_(v))(OR_(w)), (21) COR_(x); and wherein one of more C areindependently replaced by (C₃-C₃₀)-cycloalkyl, aryl,aryl-(C₁-C₃₀)-alkyl, NR_(y)R_(z), CO, O, S, Boron, halide, P and(O—CH₂—CH₂)_(B);B is an integer between 1 and 500;R_(a), R_(b), R_(c), R_(d), R_(e), R_(f), R_(h), R_(i), R_(j), R_(k),R_(m), R_(n), R_(p), R_(q), R_(s), R_(t), R_(v), R_(w), R_(x), R_(y) andR_(z) are radicals independently selected from the group consisting ofH; (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl; phenyl (C₁-C₃₀)-alkyl; and(C₃-C₈)-cycloalkyl, wherein one or more carbons are optionallysubstituted by an heteroatom selected from the group consisting of O; S;F; N; NH; P; and CO;R_(g), R_(l), R_(o), R_(r) and R_(u) are radicals independently selectedfrom the group consisting of (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl;phenyl; (C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl, wherein one or morecarbons are optionally substituted by an heteroatom selected from thegroup consisting of O; S; F; N; NH; P; and CO;R₅, R_(5′), and R_(5″) represent end-capping motif at the N-terminalposition. R₅, R_(5′) and R_(5″) are radicals independently selected fromthe group consisting of H; and (C₁-C₅₀₀)-alkyl, wherein one or more H isoptionally substituted by: (1) (C₃-C₃₀)-cycloalkyl, (2) a C-radicalderived from a ring system with 1-6 rings, each ring being independentlysaturated, partially unsaturated or aromatic, the rings being isolatedor fused and having 3-20 members each member independently selected fromthe group consisting of C, CH, CH₂, CO, N and NH, (3) OH, (4)NR_(a)R_(b), (5) ONR_(c)R_(d), (6) CN, (7) halide, (8) SH₂, (9)SR_(e)R_(f), (10) N(H)NH₂, (11) R_(g)COR_(h), (12) COOR_(i), (13)CON(R_(j))(R_(k)), (14) R_(l)N(R_(m))CON(R_(n))₂, (15) (C₁-C₃₀)-alkene,(16) (C₁-C₃₀)-alkyne, (17) N₃, (18) R_(o)CH(OR_(p))(OR_(q)), (19)R_(r)CH(SR_(s))(SR_(t)), (20) R_(u)Boron(OR_(v))(OR_(w)), (21) COR_(x);and wherein one of more C are independently replaced by(C₃-C₃₀)-cycloalkyl, aryl, aryl-(C₁-C₃₀)-alkyl, NR_(y)R_(z), CO, O, S,Boron, halide, P and (O—CH₂—CH₂)_(B);B is an integer between 1 and 500;R_(a), R_(b), R_(c), R_(d), R_(e), R_(f), R_(h), R_(i), R_(j), R_(k),R_(m), R_(n), R_(p), R_(q), R_(s), R_(t), R_(v), R_(w), R_(x), R_(y) andR_(z) are radicals independently selected from the group consisting ofH; (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl; phenyl (C₁-C₃₀)-alkyl; and(C₃-C₈)-cycloalkyl, wherein one or more carbons are optionallysubstituted by an heteroatom selected from the group consisting of O; S;F; N; NH; P; and CO;R_(g), R_(l), R_(o), R_(r) and R_(u) are radicals independently selectedfrom the group consisting of (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl;phenyl; (C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl, wherein one or morecarbons are optionally substituted by an heteroatom selected from thegroup consisting of O; S; F; N; NH; P; and CO.

In the present invention, the term “alkyl” refers to linear or branchedhydrocarbonated chain radicals, saturated, partially saturated orinsaturated. In the present invention the term “cycloalkyl” refers to acyclic hydrocarbonated radical, saturated, unsaturated or partiallysaturated or aromatic.

In an alternative embodiment, the present invention relates to thecompound of formula (I), wherein:

I₁, I₂ and I₃, are radicals independently selected from the groupconsisting of H; Deuterium; and F;

R₅, R_(5′) and R_(5″) are identical between them, and selected from thegroup consisting of H; CO—(C₁-C₂₀)-alkyl; CONH—(C₁-C₂₀)-alkyl; andpyroglutamate.

In an alternative embodiment, the present invention also relates to thecompound of formula (I) as defined in any of the above embodiments,wherein:

R₂═R_(2∝)═R_(2″), R₃═R_(3′)═R_(3″), and R₄═R_(4′)═R_(4″), and each ofthem is independently selected from the group consisting of:

X₁ and X₂ are independently selected from the group consisting of H; N;—NH₂; and Z;y and y′ are integers between 0 and 3; and y+y′=2 or 3;R₁═R_(1′)═R_(1″) is selected from the following groups:

A₁, A₂, A₃ and A₄ denote the side residues of hydrophobic amino acidsand they are selected from the following groups or combinations thereof:

L₁ is defined as in any of the above embodiments.

In a particular embodiment of any of the previous embodiments, m, m′,m″, n, n′, n″, o, o′ and o″ are integers independently selected from 0to 500, wherein at least one of them is ≥1.

In a particular embodiment of any of the above embodiments, thepolypeptidic backbone of the compound of formula (I) of the first aspectof the invention is selected from the group consisting of: arginine,ornithine, lysine, sarcosine, and serine.

In another particular embodiment of any of the above embodiments, thepolypeptidic backbone of the compound of formula (I) of the first aspectof the invention is a di-block polypeptide selected from the groupconsisting of: serine-sarcosine, serine-lysine, glutamic-serine,serine-ornithine, serine-arginine, lysine-sarcosine,sarcosine-ornithine, sarcosine-arginine, ornithine-arginine,glutamic-ornithine, glutamic-arginine, glutamic-lysine,glutamic-sarcosine, phenylalanine-glutamic, and phenylalanine-lysine,phenylalanine-ornithine, phenylalanine-arginine,phenylalanine-sarcosine, and serine-phenylalanine.

In a particular embodiment, the polypeptidic backbone of the compound offormula (I) of the first aspect of the invention is polyglutamate asdepicted below:

or its salts, solvates or isomers, wherein:m is an integer selected from 1 to 500;Z is selected from the group consisting of H; metallic counterion;inorganic counterion; and an amino acid protecting group;R₁ is selected from the following groups:

A₁, A₂, A₃ and A₄ denote the side residues of hydrophobic amino acidsand they are selected from the following groups or combinations thereof:

L₁ represents a spacer selected from the following groups:

p, q, r, s, t, u, v, and w, are integers selected from and 1 to 300respectively;and, ----- the bond which links these groups to the rest of themolecule.

In a particular embodiment, the R₁ radical of the compound of theprevious particular embodiment is selected from the following groups:

The second aspect of the invention relates a compound comprisinghomopolypeptides or random or block co-polypeptides of formula (II)below:

or its salts, solvates or isomers wherein:R₁ to R₈, I₁ to I₃, n, and o, are defined in the first embodiment;m is an integer number between 2-500;x is a number from 0.01*m to 0.5*m;R_(2′″) is a radical selected from the group consisting of:

X₁ is H;y is 0 or 1;CL is a radical selected from the group consisting of an(C₁-C₅₀₀)-alkyl, wherein one or more H is optionally substituted by: (1)(C₃-C₃₀)-cycloalkyl, (2) a C-radical derived from a ring system with 1-6rings, each ring being independently saturated, partially unsaturated oraromatic, the rings being isolated or fused and having 3-20 members eachmember independently selected from the group consisting of C, CH, CH₂,CO, N and NH, (3) OH, (4) NR_(a)R_(b), (5) ONR_(c)R_(d), (6) CN, (7)halide, (8) SH₂, (9) SR_(e)R_(f), (10) N(H)NH₂, (11) R_(g)COR_(h), (12)COOR_(i), (13) CON(R_(j))(R_(k)), (14) R_(j)N(R_(m))CON(R_(n))₂, (15)(C₁-C₃₀)-alkene, (16) (C₁-C₃₀)-alkyne, (17) N₃, (18)R_(o)CH(OR_(p))(OR_(q)), (19) R_(r)CH(SR_(s))(SR_(t)), (20)R_(u)Boron(OR_(v))(OR_(w)), (21) COR_(x); and wherein one of more C areindependently replaced by (C₃-C₃₀)-cycloalkyl, aryl,aryl-(C₁-C₃₀)-alkyl, NR_(y)R_(z), CO, O, S, Boron, halide, P and(O—CH₂—CH₂)_(B);B is an integer between 1 and 500;R_(a), R_(b), R_(c), R_(d), R_(e), R_(f), R_(h), R_(i), R_(j), R_(k),R_(m), R_(n), R_(p), R_(q), R_(s), R_(t), R_(v), R_(w), R_(x), R_(y) andR_(z) are radicals independently selected from the group consisting ofH; (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl; phenyl (C₁-C₃₀)-alkyl; and(C₃-C₈)-cycloalkyl, wherein one or more carbons are optionallysubstituted by an heteroatom selected from the group consisting of O; S;F; N; NH; P; and CO;R_(g), R_(l), R_(o), R_(r) and R_(u) are radicals independently selectedfrom the group consisting of (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl;phenyl; (C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl, wherein one or morecarbons are optionally substituted by an heteroatom selected from thegroup consisting of O; S; F; N; NH; P; and CO.

In a particular embodiment, the present invention relates to thecompound of formula (II) as defined in the second aspect of theinvention, wherein:

I₁, I₂ and I₃, are radicals independently selected from the groupconsisting of H; Deuterium; and F;

R₅ is selected from the group consisting of H; CO—(C₁-C₂₀)-alkyl;CONH—(C₁-C₂₀)-alkyl; and pyroglutamate.

In a particular embodiment the present invention relates to the compoundof formula (II) as defined in the second aspect of the invention or inthe previous embodiment, wherein:

R_(2′″) is selected from the group consisting of:

X₁ is H;y is 0 or 1;CL is selected from the group consisting of:

R₉, and R₁₁ to R₁₇ are independently selected from the group consistingof:

p and s are integers independently selected from 0 to 500;R₁₀ is selected from H and (C₁-C₄)-alkyl.

In particular embodiment of any of the embodiments of the second aspectof the invention, m is an integer between 2 and 50. In anotherparticular embodiment of any of the embodiments of the second aspect ofthe invention, p and s are independent integers selected from 0 to 50.

In a particular embodiment, the polypeptidic backbone of the compound offormula (II) of the second aspect of the invention is selected from thegroup consisting of: arginine, ornithine, lysine, sarcosine, and serine.

In another particular embodiment, the polypeptidic backbone of thecompound of formula (II) of the second aspect of the invention is adi-block polypeptide selected from the group consisting of:serine-sarcosine, serine-lysine, glutamic-serine, serine-ornithine,serine-arginine, lysine-sarcosine, sarcosine-ornithine,sarcosine-arginine, ornithine-arginine, glutamic-ornithine,glutamic-arginine, glutamic-lysine, glutamic-sarcosine,phenylalanine-glutamic, and phenylalanine-lysine,phenylalanine-ornithine, phenylalanine-arginine,phenylalanine-sarcosine, and serine-phenylalanine.

In a particular embodiment, the polypeptidic backbone of the compound offormula (II) of the second aspect of the invention is polyglutamate, asdepicted below:

or its salts, solvates or isomers wherein:m and R₁ are defined as in the particular embodiment of the first aspectof the invention;R₅ is defined as in the first aspect of the invention, and CL isselected from the following groups:

R₉, and R₁₁ to R₁₇ are independently selected from the group consistingof:

p and s are integers independently selected from 0 to 500;R₁₀ is selected from H and (C₁-C₄)-alkyl.

In a particular embodiment of the previous particular embodiment, R₁ isselected from the following groups:

The third aspect of the present invention relates to a cross-linkedself-assembled star polymer comprising a recurring unit of formula (III)below:

or its salts, solvates or isomers wherein:R₁ to R₈, I₁, to I₃, m, n and o are defined as in the first aspect ofthe invention;x is defined as in the second aspect of the invention;R_(2′″) is selected from the group consisting of:

X₁ and y are defined as in the second aspect of the invention;CL₁ is defined as CL the second aspect of the invention.

In a particular embodiment, the present invention relates to thecross-linked self-assembled star polymer of formula (III), as defined inthe third aspect of the invention wherein:

I₁, I₂, and I₃, are radicals independently selected from the groupconsisting of H; Deuterium; and F;

R₅ is selected from the group consisting of H; CO—(C₁-C₂₀)-alkyl;CON(H)—(C₁-C₂₀)-alkyl; and pyroglutamate.

In another particular embodiment, the present invention relates to thecross-linked self-assembled star polymer according to the third aspectof the invention or to the previous preferred embodiment, wherein:

each R₂, R₃, and R₄ is independently selected from the group consistingof:

X₁ and X₂ are defined as in the first aspect of the invention;y and y′ are defined as in the first aspect of the invention;R₁ is selected from the following groups:

A₁, A₂, A₃ and A₄ denote the side residues of hydrophobic amino acidsand they are selected from the following groups or combinations thereof:

L₁ represents a spacer selected from the following groups:

p, q, r, s, t, u, v, and w, are integers selected from and 1 to 300respectively;and, ----- the bond which links these groups to the rest of themolecule.

In another particular embodiment, the present invention relates to theself-assembled star polymer according to the third embodiment of theinvention or any of the two previous embodiments, wherein:

R_(2′″) is selected from the group consisting of:

CL₁ is selected from the group consisting of:

R₉, and R₁₁ to R₁₇ are selected from the group consisting of:

p and s are integers independently selected from 0 to 500;R₁₀ is selected from H and (C₁-C₄)-alkyl.

In a particular embodiment, the polypeptidic backbone of thecross-linked self-assembled star polymer comprising a recurring unit offormula (III) is selected from the group consisting of: arginine,ornithine, lysine, sarcosine and serine.

In another particular embodiment, the polypeptidic backbone of thecross-linked self-assembled star polymer comprising a recurring unit offormula (III) is a di-block polypeptide selected from the groupconsisting of: serine-sarcosine, serine-lysine, glutamic-serine,serine-ornithine, serine-arginine, lysine-sarcosine,sarcosine-ornithine, sarcosine-arginine, ornithine-arginine,glutamic-ornithine, glutamic-arginine, glutamic-lysine,glutamic-sarcosine, phenylalanine-glutamic, and phenylalanine-lysine,phenylalanine-ornithine, phenylalanine-arginine,phenylalanine-sarcosine, and serine-phenylalanine.

In a particular embodiment, the polypeptidic backbone of thecross-linked self-assembled star polymer comprising a recurring unit offormula (III) is polyglutamate as depicted below:

wherein:R₁, R₅, m, and x are defined as in the second aspect of the inventionwhere the polypeptidic backbone is polyglutamate;CL₁ is selected from the following groups:

R₉ to R₁₇ are defined as in the first particular embodiment of thesecond aspect of the invention.

In another particular embodiment, the R₁ radical of the cross-linkedself-assembled star polymer of the previous particular embodiment isselected from the following groups:

The compounds and the cross-linked self-assembled star polymer of thepresent invention as defined in any of the previous embodiments oraspects of the invention may include isomers, depending on the presenceof multiple bonds (for example, Z, E), including optical isomers orenantiomers, depending on the presence of chiral centers. The individualisomers, enantiomers or diastereoisomers, and the mixtures thereof, fallwithin the scope of the present invention. The individual enantiomers ordiastereoisomers, and the mixtures thereof, may be separated by means ofany conventional technique well known to the person skilled in the art.

The compounds and the cross-linked self-assembled star polymer of thepresent invention may be in crystalline form as free ones or assolvates, and both forms are intended to be included within the scope ofthe present invention. In this regard, the term “solvate”, as usedherein, includes both pharmaceutically acceptable solvates, i.e.solvates of the compound with the formula (I), or (II) or thecross-linked self-assembled star polymer (III) that may be used in thepreparation of a medicament, and pharmaceutically unacceptable solvates,which may be useful in the preparation of pharmaceutically acceptablesolvates or salts. The nature of the pharmaceutically acceptable solvateis not critical, provided that it is pharmaceutically acceptable. In aparticular embodiment, the solvate is a hydrate. The solvates may beobtained by conventional solvation methods that are well-known topersons skilled in the art. Except as otherwise specified, the compoundsof the present invention also include compounds that differ only in thepresence of one or more isotope-enriched atoms. Examples ofisotope-enriched atoms, without limitation, are deuterium, tritium, ¹³Cor ¹⁴C, or a nitrogen atom enriched in ¹⁵N.

A fourth aspect of the invention relates to a conjugate comprising thecompounds of formula (I) or (II) as previously defined, or theself-assembled star polymer of formula (III) as previously defined, andat least an active agent linked to the compound or the self-assembledstar polymer. The at least active agent(s) can be covalently bounddirectly or by one or more linkers, or alternatively the at least activeagent(s) can be non-covalently bound to the compound or theself-assembled star polymer. In a preferred embodiment, the at leastactive agent is (are) covalently linked to the polypeptidic backbonethrough the amino acid side residue via amide, ester, anhydride bondingor through a linker that include one or more functional groups,including without limitation, alkynes, azides, reactive disulfides,maleimides, hydrazide, hydrazones, Schiff bases, acetal, aldehydes,carbamates, and reactive esters. In an alternative embodiment thecovalent link is a bioresponsive one. The expression “bioresponsive one”refers to a chemical link cleavable under specific physiological orexternal triggers (for example, and without limitation, pH, reactiveoxygen species, reductive environment, specific enzymes, glucose, light,temperature, etc.).

In the present invention the expression “active agent” refers to anactive ingredient and an imaging agent.

Preferably, the active ingredient is selected from small agents (i.e.pharmaceutical active ingredients or drugs) to biomolecules (i.e.peptides, (apolipo)proteins, antibodies, Fab or fragmentantigen-binding, and nucleic acids). Examples of active ingredientinclude, without limitation, antibody, antigen,(arginine-glycine-aspartate (RGD)) peptide, oligosaccharide,bisphosphonate, aptamer, polysaccharide, hyaluronic, chondroitinsulphate, double stranded oligonucleotide (DNA), siRNA, fibronectin, andfolate.

In preferred embodiment, the active ingredient is selected from thegroup consisting of anticancer agent, antimetastatic, agentanti-inflammatory agent, antioxidant, neuroprotective agent,immunostimulant agent, agent capable to trigger tissue repair and/orregeneration, antioxidants, antiapoptotic, proapoptotic,anti-amyloidotic agent, and plaque/protein aggregates disrupting agents.

In a still preferred embodiment, the active ingredient is selected fromthe group consisting of: vincristine, vinblastine, amiloride,chloroquine, blafiomycyn, fasudil, bisphosphonate, primaquine,meclofenamate, tonabersat, disulfiram, cyclophosphamide, paclitaxel,dendrotoxin, doxorubicine, methotrexate, epirubicine, dinaciclib,buparlisib, palbociclib, veliparib, megestrol, examestane, goserelin,tamoxifen, fulvestrant, trastuzumab, lapatinib, pertuzumab, selegiline,rasagiline, ladostigilM30, curcumin, demethoxycurcumin, andbisdemethoxycurcumin.

In the present invention the expression, “imaging agent” refers to anysubstance that is used as a label, or that enhances specific structuresin any imaging technique. An imaging agent, hence, includes opticalimaging agent, magnetic resonance imaging agent, radioisotope, andcontrast agent. Examples, without limitation, of optical imaging agentare acridine dye, a coumarin dye, a rhodamine dye, a xanthene dye, acyanine dye, and a pyrene dye, Texas Red, Alexa Fluor® dye, BODIPY® dye,Fluorescein, Oregon Green® dye, and Rhodamine Green™ dye, which arecommercially available or readily prepared by methods known to thoseskilled in the art. Examples of imaging agents appropriate for thepresent invention include, but are not limited to, transition metals andradioactive transition metals chelated to chelating agents, for instanceDTPA (diethylene triamine pentaacetic acid), DOTA(1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid) and NOTA(1,4,7-Triazacyclononane-1,4,7-triacetic acid).

In an alternative embodiment, the conjugate defined in the fourth aspectof the invention or any embodiment thereto, comprises an amount of theat least an active agent in the range of 1 to 70% w/w based on the massratio of the active agent to the conjugate. In a preferred embodiment,the range is of 1 to 50% w/w. In a still more preferred embodiment, theconjugate comprises an amount of the agent in the range of 1 to 25% w/w.

A fifth aspect of the invention relates to a pharmaceutical, diagnosticor theranostic composition comprising at least one conjugate as definedin the fourth aspect of the invention, or any embodiment together withone or more appropriate pharmaceutical or diagnostically acceptableexcipients.

A sixth aspect of the invention relates the conjugate for use as amedicament, in diagnostics or a combination of both (theranostics).

This aspect could also be formulated as a method for the prophylaxisand/or treatment of a disease which comprises administering to mammalsin need of such treatment or diagnostic, an effective amount of any ofthe conjugates of the present invention, together with one or moreappropriate pharmaceutically acceptable excipients and/or carriers. Thisaspect could also be formulated as a method for the diagnosis of adisease in an isolated sample of a subject, the method comprisesadministering to said subject an effective amount of the any of theconjugate having one or more imaging agents as defined above to theisolated sample of the subject. The detection of these imaging agentscan be carried out by well-known techniques such as imaging diagnostictechniques. Examples of imaging diagnostic techniques suitable for thepresent invention include, but not limited to, are magnetic resonanceimaging (MRI), X-ray, positron emission tomography (PET), single-photonemission computed tomography (SPECT), fluorescence microscopy, and invivo fluorescence.

In a particular embodiment of this aspect, the conjugate for use in theprevention and/or treatment of neurodegenerative disorder, neurologicaldisease, cancer, infectious disease, disorder related to aging,neuro-inflammation, demyelinating disorder, multiple sclerosis, ischemicdisorder, ischemia-reperfusion damage, amyloydotic disease,cardiomyopathy, spinal cord injury, immune disorder, inflammatorydisorders, rare disease, wound healing and lysosomal storage disease.

The neurodegenerative diseases may be selected from the list thatcomprises, without being limited thereto, Alzheimer's disease,Parkinson's disease, amyotrophic lateral sclerosis, cerebral ischaemia,post-encephalitic Parkinsonisms, dystonias, Tourette syndrome, periodiclimb movement pathologies, restless legs syndrome, attention deficithyperactivity disorders, Huntington's disease, progressive supranuclearpalsy, Pick's disease, fronto-temporal dementia and neuromusculardiseases.

The compounds described in the present invention, their pharmaceuticallyacceptable salts and solvates, and the pharmaceutical compositionscontaining them may be used jointly with other, additional drugs, toprovide combined therapy. Said additional drugs may be a part of thesame pharmaceutical composition or, alternatively, may be provided inthe form of a separate composition for simultaneous or non-simultaneousadministration with the pharmaceutical composition comprising a compoundwith the formula (I) or the formula (II), or the cross-linkedself-assembled star polymer a pharmaceutically acceptable salt,stereoisomer or solvate thereof.

The compounds with the formula (I), formula (II) or cross-linkedself-assembled star polymer of formula (III) designed for therapeuticuse are prepared in solid form or aqueous suspension, in apharmaceutically acceptable diluent. These preparations may beadministered by any appropriate administration route, for which reasonsaid preparation will be formulated in the adequate pharmaceutical formfor the selected administration route. In a particular embodiment,administration of the compound of formula (I) or (II), or cross-linkedself-assembled star polymer with the formula (III) provided by thisinvention is performed by oral, topical, rectal or parenteral route(including subcutaneous, intraperitoneal, intradermal, intramuscular,intravenous route, etc.). A review of the different pharmaceutical formsfor the administration of medicaments and the necessary excipients toobtain them may be found, for example, in “Tratado de FarmaciaGalénica”, C. Faulí i Trillo, 1993, Luzán 5, S. A. Ediciones, Madrid, orother habitual or similar ones in the Spanish Pharmacopeia and in theUnited States.

In an eight aspect, the present invention relates to a process for thesynthesis of the compound of formula (I) of the first aspect of theinvention or any embodiment thereto, the process comprising:

-   -   (1) reacting an amine or tetrafluoroborate or trifluoroacetate        ammonium salt form of initiator of formula (IV) below

-   -   -   with an appropriate N-carboxyanhydride (NCA); alternatively,            reacting the amine or tetrafluoroborate or trifluoroacetate            ammonium salt form of initiator of step (1) with an            appropriate N-carboxyanhydrides in a sequential manner to            obtain a block co-polymer; alternatively, reacting the amine            or tetrafluoroborate or trifluoroacetate ammonium salt form            of initiator of step (1) with an appropriate NCA mixture in            a statistical manner to obtain random co-polymers;

    -   (2) optionally, reacting the amine group at the N-terminal        position with an amine reactive group to introduce R₅, R_(5′)        and/or R_(5″);

    -   (3) optionally, orthogonally removing amino acid side chain        protecting groups;

    -   (4) purifying the product obtained in step (1), (2) or (3),        optionally by fractionation, precipitation, ultrafiltration,        dialysis, size-exclusion chromatography or tangential flow        filtration.

Step (1) above may include: a) ring opening polymerization of aminoacids N-carboxyanydride (NCA) monomer by reacting the amine ortetrafluoroborate or trifluoroacetate ammonium salt form of initiator offormula (IV) above with the selected NCA, wherein the ratiomonomer/initiator allow to control the degree of polymerization (DP); b)a sequential polymerization, wherein block co-polypeptides are preparedfollowing the polymerization reaction a) in a sequential manner,allowing the first NCA monomer to be consumed before adding the monomerto build the following polypeptidic block; or c) a statisticalpolymerization a) wherein random copolypeptides are prepared followingthe polymerization reaction a) in a statistical manner, mixing all theNCA monomers before starting the polymerization by the addition of anamine or tetrafluoroborate or trifluoroacetate ammonium salt form ofinitiator.

Step (2) above corresponds to the end-capping, wherein the amine groupat the N-terminal position is reacted with an amine reactive group tointroduce R₅, R_(5′) or R_(5″).

Step (3) above corresponds to the deprotection, wherein amino acid sidechains are removed orthogonally depending on the protecting grouppresent at Z.

The process for the synthesis of the compound of formula (II) of thesecond aspect of the invention or any embodiment thereto, the processfurther comprising:

-   -   (5) introducing the CL groups at reactive amino acid side chain,        at the appropriate molar ratio;    -   (6) purifying the product obtained in step (5), optionally by        fractionation, precipitation, ultrafiltration, dialysis, size        exclusion chromatography or tangential flow filtration.

Step (5) above corresponds to the postpolymerization modification ofamino acid side chain, wherein the required modification is introducedat the reactive amino acid side chain at the desired molar ratio tointroduce CL groups.

The process for the synthesis of the cross-linked self-assembled starpolymer of formula (III) of the third aspect of the invention or anyembodiment thereto, the process further comprising:

-   -   (7) reacting the CL groups of the self-assembled compounds of        formula (II) forming nanometric assemblies, to covalently        cross-link the self-assembled star polymers;    -   (8) purifying the product obtained in step (7) optionally by        fractionation, precipitation, ultrafiltration, dialysis, size        exclusion chromatography or tangential flow filtration.

Step (7) above corresponds to the self-assembly and covalentcross-linking step, wherein compounds of formula (II) are self-assembledunder the appropriate conditions depending on their nature to formnanometric assemblies. Then CL groups are reacted to covalentlycross-link the self-assembled star polymers.

In another particular embodiment, the compound of formula (I) is acompound of formula (IA):

or its salts, solvates or isomers, wherein m is an integer selected from1 to 500, Z is selected from H, lineal or chain alkyl C₁-C₁₀, aryl orSiRR′ being R and R′ alkyl C₁-C₆ or a metallic or organic cation,R₁ is selected from the following groups:

wherein A₁, A₂, A₃ and A₄ denotes the side residues of hydrophobic aminoacids and they are selected from the following groups or combinationsthereof:

and L₁ represents a spacer selected from the following groups:

being p, q, r, s, t, u, v and w integers selected from 0 to 10 and 1 to300 respectively, and ------- the bond which links these groups to therest of the molecule.

In a preferred embodiment, R₁ of formula (IA) is selected from thefollowing groups:

In another embodiment, the compound of formula (IA) is a compound offormula (IIA):

or its salts, solvates or isomers wherein m and R₁ are defined as in theparticular embodiment of the invention corresponding to a compound offormula (IA) as defined for the first time above, x is and CL isselected from the following groups:

wherein R₉ to R₁₇ are selected from the following groups:

being p an integer selected from 0 to 10 and s an integer selected from1 to 300 respectively, and R₁₀ is selected from a hydrogen or an alkylC₁-C₄,and wherein the described substituent is also present in the positionsmarked with R.

In a preferred embodiment of the compound of formula (IIA), R₁ isselected from the following groups:

Another aspect of the invention is referred to a polymer comprising arecurring unit of formula (IIIA):

wherein R₁, m and x are defined as in the particular embodiment of theinvention corresponding to a compound of formula (IA) as defined for thefirst time above and CL₁ is selected from the following groups:

wherein R₉ to R₁₇ are defined as above.

In a preferred embodiment of the compound of formula (IIIA), R₁ isselected from the following groups:

Another aspect of the invention, is a conjugate comprising the compoundof formula (IIA) or the compound of formula (IIIA) and at least an agentwhich is covalently linked to the compound through the COOH surfacegroups of the compound of formula (IIIA) or through a spacer, preferablythe agent is selected from the group consisting of a drug, a targetingagent, an optical imaging agent, a magnetic resonance imaging agent anda stabilizing agent, more preferably the conjugate of previous claimwherein the agent is a drug selected from selegiline, rasagiline,ladostigilM30, curcumin, demethoxycurcumin, bisdemethoxycurcumin.

Preferably, in the conjugate described above the conjugate comprises anamount of the agent in the range of 1% to 50% (weight/weight) based onthe mass ratio of the agent to the conjugate.

Another aspect refers to a pharmaceutical composition comprising atleast one conjugate as described above and at least a pharmaceuticalexcipient.

Another aspect refers to the conjugate described above for use as amedicament.

Another aspect refers to the conjugate as described above for use in theprevention or treatment of a neurodegenerative or neurological diseaseor cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skilledin the art to which this invention belongs. Methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention. Throughout the description and claimsthe word “comprise” and its variations are not intended to exclude othertechnical features, additives, components, or steps. Additional objects,advantages and features of the invention will become apparent to thoseskilled in the art upon examination of the description or may be learnedby practice of the invention. The following examples, drawings areprovided by way of illustration and are not intended to be limiting ofthe present invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1. a) Selected GPCs from St-Poly-(gamma-benzyl-L-glutamate) (PBLG)(compounds of formula (I)) of different molecular weights in DMF (1%LiBr) at 8 mg·mL⁻¹. b) CD of St-PBLG (compounds of formula (I)) at 20°C. in HFIP at 0.1 mg·mL⁻¹.

FIG. 2. a) Selected GPCs in DMF (1% LiBr) at 8 mg·mL⁻¹ and b) Selected¹H-NMR and respective signal assignment in deuterated TFA of St-PTLL:Star-Poly(epsilon-trifluoroacetate-L-Lysine), St-PBLS:Star-Poly(Benzyl-L-Serine), St-PSAR: St-Poly(Sarcosine), St-PBLG:St-Poly(gamma-benzyl-L-glutamate) (compounds of formula (I)). * R₁ is anethyl.

FIG. 3. a) Selected GPCs in DMF (1% LiBr) at 8 mg·mL⁻¹ and b) Selected¹H-NMR and respective signal assignment in deuterated TFA of St-PSAR:St-Poly(Sarcosine), St-PBLG: St-Poly-(gamma-benzyl-L-glutamate),St-P(BLG-co-SAR): St-poly(gamma-benzyl-L-glutamate-co-sarcosine) randomcopolymer, St-P(BLG-b-SAR):St-poly(gamma-benzyl-L-glutamate-block-sarcosine) block copolymer(compounds of formula (I)). * R₁ is an ethyl.

FIG. 4. a) ¹H-NMR of St-PGAs (compounds formula (I)) of differentmolecular weights in D₂O. The small square is surrounding the benzylcore signals. b) CD of St-PGAs (compounds formula (I)) in PBS at 37° C.at 1 mg/mL showing typical random coil conformation of PGA chains.

FIG. 5. a) ¹H-NMR of (compounds formula (I)), St-P(BLG-co-SAR):St-poly(gamma-benzyl-L-glutamate-co-sarcosine) random copolymer,St-P(BLG-b-SAR): St-poly(gamma-benzyl-L-glutamate-block-sarcosine) blockcopolymer in D₂O. * R₁ is an ethyl.

FIG. 6. a) ¹H-NMR of (compounds formula (II)), St-PGAs with differentfunctionalities in D₂O.

FIG. 7. SANS data plotting of various St-PGAs (compound of formula (I))at 10 mg·mL⁻¹ in PBS buffer at pH=7.4.

FIG. 8. Size-concentration dependence analysis by DLS in PBS buffer atpH 7.4. a) Mean Count Rate (MCR) vs. concentration of star-shapedpolymer with ethyl-based initiator, DP 180 as example (compound offormula (I)). b) Mean Count Rate vs. concentration of linear PGA polymerDP 150 as example.

FIG. 9. Schematic representation of the self-assembly process followedby star shaped polyglutamates (compounds of formula (I) and (II))studied according to DLS and SANS data interpretation.

FIG. 10. ²H NMR spectra of the D-labeled initiator with correspondingpeaks assignments. a) N-Boc-protected initiator in H₂O+3 μL Acetone-d6at 300 MHz. b) BF₄ initiator in H₂O+3 μL Acetone at 500 MHz.

FIG. 11. SANS contrast experiments with D-labeled core in D₂O (outer Hmolecular organization determination) and H₂O (D-labeled core molecularorganization determination), at 10 mg·mL⁻¹ and 20° C. of DeuteratedSt-PGA (Compound of formula (I)).

FIG. 12. Cryo-TEM micrographs from St-PGA of a sample (Compound offormula (I)) prepared in ddH₂O at 1 mg·mL⁻¹

FIG. 13. Ionic strength effect on size of St-PGA (Compound of formula(I)) at 2 mg·mL⁻¹ and at 37° C. represented by the changes suffered inscattered intensity (MCR) (left) and in R_(h) by number (right) uponaddition of increasing amount of different salts.

FIG. 14. a) Temperature effect on size of St-PGA (Compound of formula(I)) at 10 mg·mL⁻¹. b) Size-concentration dependence of St-PGA (Compoundof formula (I)) at 37° C.

FIG. 15. Mean count rate (MCR) vs. increasing concentrations plotting ofa) alkyne modified St-PGAs (Compound of formula (II)) with differentdegrees of functionalization; b) azide modified St-PGAs with differentdegrees of functionalization (Compound of formula (II)); c) linear PGAalkyne modified as negative control and d)

FIG. 16. Cryo-TEM micrographs of modified star polyglutamates (Compoundof formula (II)) at 1 mg·mL⁻¹ in ddH₂O; a) St-EG(2)N₃(5); b)St-prop(10).

FIG. 17. Co-assembly study by DLS. Graphs showing CAC determination forSt-EG(2)N₃(5), (Compound of formula (II)), in the presence of constantconcentration of St-prop(10), (Compound of formula (II)), (a) and viceversa (b).

FIG. 18. Co-assembly studies through DOSY NMR of (Compounds of formula(II)), graphs obtained by fitting the intensities of the arrayed DOSYspectra into Stejskal-Tanner equation and the calculated diffusioncoefficients (D).

FIG. 19. 2D NOESY spectra showing NOE correlation of propargyl andethylene glycol protons of a mixture containing 2 mg·mL⁻¹ of eachcompounds of formula (II): St-prop(10) and St-EG(2)N₃(5).

FIG. 20. Schematic representation of covalent capture of co-assembledstar-shaped polymers bearing orthogonal functionalities (Compounds offormula (II) and (III)) exemplified for PGA.

FIG. 21. Schematic representation of covalent capture of co-assembledstar-shaped polymers bearing orthogonal functionalities (Compounds offormula (II)) (specific case of alkynes and azides) through CuAAC clickchemistry yielding compounds of formula (III).

FIG. 22. ¹H-NMR in D₂O of compounds of formula (III) obtained bydifferent cross-linking chemistries as example, and compared with theirprecursors compounds of formula (II). a) Cross-linked system (formula(III)) by CuAAC chemistry and St-prop(10) and St-EG(2)N3 (5), (compoundof formula (II)). b) Cross-linked system (formula (III)) by di-thiochemistry and St-PD(30) (compound of formula (II)).

FIG. 23. Comparison of both measured systems (compounds of formula IIphysically mixed, and compound of formula III) in terms of size againstconcentration. All values correspond to measurements in PBS 7.4 and arerepresented in number.

FIG. 24. Cryo-TEM pictures at 2 mg·mL⁻¹ of compounds of formula (III).

FIG. 25. In vitro evaluation of the newly synthesized St-PGA ((compoundsformula (I)) carriers. a) Example of degradation profile by cathepsin Bmonitored by GPC in PBS at 3 mg/mL and at different time points. b)Toxicity assay against SHSY5Y cell line of different St-PGAs (compoundsformula (I)) measure by MTS assay at 72 hours post-treatments. c)Toxicity assay against HUVEC cell line of different PGA basedarchitectures measure by MTS assay at 72 hours post-treatments. DB:Di-block PEG₄₂PGA₂₀₀; PGA: linear PGA₂₅₀; STAR: St-PGA₂₅₀

FIG. 26. In vitro evaluation of the newly synthesized carriers. a)Uptake study by flow cytometry of a star-shaped fluorescently labeledPGA (compounds formula (I)) in SHSY5Y cell line. Experiment at 4° C.excludes the energy dependent mechanisms. The different among the uptakeprofile at 4 and 37 degrees (so-called “Energy-dependent” uptake) isalso represented. CAF: Cell associated fluorescence=% positivecells*mean fluorescence/100. The CAF represented corresponds to thedifference between CAF obtained with treated cells and CAF fromuntreated control cells. b) Confocal image of the uptake at 2 hourspost-treatment of a star-shaped PGA in SHSY5Y cell line following apulse-chase experiment, with co-localization histogram. Co-localizationwith the lysosomal marker Lysotracker Red was observed.

FIG. 27. Uptake study of St-PGA ((compounds formula (I)) in comparisonwith linear PGA of similar MW (around 250 GAU). a) CAF of both polymersover time. b) CAF of both polymers at 5 hour time point showingsignificant differences when statistics was performed using one-wayANOVA. p*<0.05. c) % of positive (+) cells to Oregon Green (OG)fluorescence, comparison of both polymers at 5 hour time point showingas expected statistical differences. p*<0.05. d) % of positive cellsrepresentation comparing both polymers together with the control used(cell autofluorescence).

FIG. 28. ¹H-NMR spectra (D₂O) of St-DO3A-tBu and St-DO3A (conjugates ofcompounds of formula (I)).

FIG. 29. % Activity measured after ¹¹¹In labeling and purification bySEC of St-DO3A (compound of formula (I)).

FIG. 30. St-DO₃A-¹¹¹In biodistribution. Data expressed as normalized %ID per gram of tissue at different time points.

FIG. 31. Normalized data of radioactivity signal of each organ respectthe injected dose (ID) per gram of tissue, of St-PGA (compound offormula (I) compared with it linear counterpart of similar MW.

FIG. 32. Cell viability assay of 3 different St-Click architectures(compounds of formula (III)) against SHSY5Y cell line up to 3 mg·mL⁻¹,72 hours of treatments (n>3, mean±SEM).

FIG. 33. a) Uptake kinetics against SHSY5Y cell line ofSt-Click-OG-labeled (compound of formula (III)) polymer at differenttime points and different temperatures (4° C. for binding, 37° C. fortotal uptake). b) CAF representing the energy-dependent fraction ofuptake, comparing the three architectures over time. n>3, mean±SEM.

FIG. 34. Confocal image of the uptake at 2 hours post-treatment ofOG-labeled St-Click compound of formula (III)) in SHSY5Y cell linefollowing a pulse-chase experiment. Co-localization with Lysotracker Redwas observed.

FIG. 35. Schematic representation of the synthetic route followed forsurface modification of the covalently captured star-shaped PGAs(compounds of formula (III)) to reach the dual probes. a) 1) DMTMM.Cl,2) DO3AtBu-NH₂ in ddH₂O, r.t. 24 h. b) and f) 1) DMTMM.Cl, 2) Cy5.5(6S-IDCC) in ddH₂O, r.t. 24 h. c) and g) TFA:TIPS:ddH₂O (95:2.5:2.5),r.t. 3 h. d) and i) GdCl³⁺ in PBS 0.1 M 7.4, r.t. 5 h. e) 1) DMTMM.Cl,2) cysteamine-SS2TP in ddH₂O, r.t. 24 h. h) ANG in HEPES buffer 7.4,r.t. 16 h. *Yellow disc: DO3A, *Purple disc: DO3A-Gd3+*Blue star: Cy5.5,*Green arrow: Angiopep-2

FIG. 36. Z-potential obtained at 20° C. from clicked structures(compounds of formula (III)) at 1 mg·mL⁻¹ in ddH₂O, before and after thesubsequent surface modifications.

FIG. 37. TEM micrographs of a) St-Click-DO3A-Gd-Cy5.5, and b)St-Click-DO3A-Gd-Cy5.5-ANG (compounds of formula (III))

FIG. 38. % ID normalized by pixel area of non-targeted (a) and targeted(b) Cy5.5 labeled clicked architectures (compounds of formula (III)).

FIG. 39. Biodistribution by optical imaging at different time points oftargeted and non-targeted clicked architectures (compounds of formula(III)). Time course experiment. Error bars are not included for clarityreasons.

FIG. 40. Cell viability of BDMC derivatives (compounds of formula (I, orIII)) against SHSY5Y cell line. 72 hours MTS assay. n>3, mean±SEM.

FIG. 41. Drug release profiles at different pH (5.0, 6.5 and 7.4) ofSt-Click-BDMC (4 wt %), (compound of formula (III)). Time courseexperiments were done per triplicate. n>3, mean±SEM.

FIG. 42. a) Schematic representation of ThT fluorescence changes uponprotein fibrillization. b) Pictures of HEWL unimers and HEWL fibrilsupon heating at 60° C. and vigorous stirring during 24 h, pH 2.0.

FIG. 43. ThT fluorescence intensity changes upon time in HEWL samplesincubated with different BDMC conjugates (compound of formula (I orIII)) at a) 10 μM BDMC-eq. and b) 50 μM BDMC-eq. n>3, mean±SEM.

FIG. 44. TEM pictures obtained from HEWL incubated samples within thedifferent BDMC polyglutamate derivatives (compound of formula (I, orIII)) at 10 μM BDMC-eq. (c-e) in comparison with a) Control PBS and b)Free BDMC 10 μM.

FIG. 45. Changes in density (nuclei/1000 μm²) of PI stained nuclei inpyramidal layer of CA1 region of hippocampal organotypic culturescomparing control cultures treated with vehicle and cultures treatedwith different concentrations of St-Click-BDMC, (compound of formula(III)) (0.005, 0.05, 0.2 and 0.5 μM drug-eq.). Asterisk indicatestatistically significant differences after ANOVA analyses followedBonferroni's post hoc tests. n>3, mean±SEM.

FIG. 46. Changes in density (nuclei/1000 μm²) of PI stained nuclei inpyramidal layer of CA1 region of hippocampal organotypic culturescomparing control cultures treated with vehicle (No polymer/No Aβ),cultures pretreated with different concentrations of polymer conjugate(compound of formula (III)) (0.05 μM drug-eq. (Polymer 0.05/No Aβ) and0.2 μM drug-eq. (Polymer 0.2/No Aβ), exposed only to Aβ₁₋₄₂ peptide (Nopolymer/Aβ) or exposed to Aβ₁₋₄₂ and pretreated with differentconcentrations of polymer conjugate (0.05 μM drug-eq. (Polymer 0.05/Aβ)and 0.2 μM drug eq. (Polymer 0.2/Aβ). Blue asterisk in bars indicatestatistically significant differences from control group and redasterisk indicate statistically significant differences from culturesexposed only to Aβ₁₋₄₂ peptide (No polymer/Aβ), after ANOVA analysesfollowed by Bonferroni's post hoc tests. n>3, mean±SEM.

FIG. 47. Experimental design from the treatments with compound offormula (III) performed in ArcAbeta model together with the animalweight registration as a proof of treatment safety.

FIG. 48. ¹H-NMR in D₂O of a) St-PGA-PD(22%)-Hyd(8.8%) andSt-PGA-PD(22%)-Hyd-Boc(8.8%) with assignations, and b)St-PGA-PD(22%)-Cl-Hyd(8.6%) and St-PGA-PD(22%)-Cl-Hyd-Boc(8.6%) withassignations. R′ represents the binding point to the recurring formula(III).

FIG. 49. Cell viability against 4T1 breast cancer cells incubated withfree DOX and St-PGA-PD(22%)-Cl-Hyd(8.4%)-Dox(2.5%), compound of formula(III).

FIG. 50. Drug release studies at pH5 and pH7.4 ofSt-PGA-PD(22%)-Cl-Hyd(8.4%)-Dox(2.5%), compound of formula (III).

EXAMPLES Example 1: Synthesis of Compounds of Formula (I)

To synthesize compounds of formula I, first, the 3-arm star initiatorwas obtained in 2-4 steps. Such initiator was used to polymerizegamma-benzyl L-glutamate N-carboxyanhydride monomer, to yield the starpolymer benzyl protected (St-PBLG). The benzyl groups were removed toyield St-PGA.

1.1. Example of Synthesis of 3-Arm Star Initiators a) Synthesis of1,3,5-tri-tert-butyl ((benzenetricarbonyltris(azanediyl))tris(ethane-2,1-diyl))tricarbamate

In a two-neck round bottom flask fitted with a stirrer bar, and a N₂inlet and outlet, 500 mg of 1,3,5-benzenetricarbonyl trichloride (1.88mmol, 1 equivalent (eq.)) was dissolved in 12 mL of anhydrous THF.N,N′,N″-diisopropylethylendiamine (DIEA) (803.31 mg, 6.22 mmol, 3.3 eq.)was added to the reaction mixture followed by drop-wise addition ofN-tert-Butoxycarbonyl-ethylendiamine (N-Boc-ethylenediamine) (1.34 g,6.22 mmol, 3.3 eq.) over a period of 10 min. The reaction was then leftto proceed for 2 hours. After that time, the solvent was completelyremoved under vacuum. The product was re-dissolved in chloroform andwashed 3 times with deionized water (ddH₂O), and 3 times with acidicwater (pH-3). Finally, the organic phase was isolated under vacuum andthe product was recrystallized 3 times from THF/Methanol/Hexane yieldinga white crystalline solid. The product was then dried under high vacuumand stored at −20° C.

Yield: 82%. ¹H NMR (300 MHz, DMSO) δ 8.68-8.65 (m, 3H), 8.41 (s, 3H),6.92-6.88 (m, 3H), 3.34-3.31 (m, 6H), 3.16-3.13 (m, 6H), 1.37 (s, 27H).¹³C NMR (75 MHz, CDCl₃) δ 166.80 (C═O), 156.84 (C═O), 134.58 (C_(Ar)quaternary), 128.47 (CH_(Ar)), 79.57 (C quaternary), 40.93 (CH₂), 40.43(CH₂), 28.45 (CH₃).

b) Synthesis of 1,3,5-(benzenetricarbonyltris(azanediyl))triethanamoniumBF₄ Salt

In a round bottom flask fitted with a stirrer bar and a stopper, 200 mgof 1,3,5-Tri-tert-butyl ((benzenetricarbonyltris(azanediyl))tris(ethane-2,1-diyl))tricarbamate (0.33 mmol, 1 eq.) was dissolved indichloromethane. Afterwards, 3.3 eq. of tetrafluoroboric acid diethylether complex, HBF₄.Et₂O, (179 mg, 150 μL), was added to the solutionleading to the instantaneous formation of a white solid. The precipitatewas filtered off and recrystallized three times fromTHF/methanol/hexane. The product was then dried under high vacuum andstored at −20° C.

Yield: 98%. ¹H NMR (300 MHz, D₂O) δ 8.32 (s, 3H), 3.72-3.68 (m, 6H)3.25-3.21 (m, 6H). ¹³C NMR (75 MHz, D₂O) δ 169.45 (C═O), 134.38 (C_(Ar)quaternary), 129.36 (C_(Ar)), 39.23 (CH₂), 37.52 (CH₂). 19F-NMR: −150.48(BF₄). MALDI-TOF: 337.1709 [M⁺¹]

Initiators used for the preparation of polypeptides in table 1 aresynthesized following analogous procedures as the ones described above.For L-phenylalanine containing initiators the synthetic route and their¹H NMR signals are summarized below. For clarity only one of the threesubstituents at the 1,3,5-benzenetricarboxamide motif is shown:

Procedure: 1,3,5-benzenetricarboxylic acid (1 eq.) was dissolved inmethanol and DMTMM chloride was added (3.9 eq.). After 10 minutessolution of N-Boc-N′-(L-phenylalaninyl) ehhylenediamine (3.9 eq.) inmethanol was added. Reaction allowed to proceed for 48 h. Product wasprecipitated in water, filtered and washed with water. The product wasfreeze-dried.

Procedure: In a round-bottom flask fitted with a stirrer compound (1eq.) was suspended in 1 ml of methanol. 4M HCl solution in dioxane wasadded (9 eq.). Reaction allowed to proceed for 2 hours. Mixture wasprecipitated in diethyl ether, washed and redissolved and treated withNaOH (till pH 10). Precipitate was filtered, washed with water andfreeze-dried.

1H-NMR (DMSO-d6): 2.54 (dt, 1H), 2.94-3.14 (m, 7H), 4.72 (dt, 1H),7.12-7.35 (m, 5H), 8.07 (t, 1H), 8.23 (s, 1H), 8.75 (d, 1H).

Procedure: 1,3,5-benzenetricarboxylic acid (1 eq.) was dissolved inmethanol and DMTMM chloride was added (3.9 eq.). After 10 minutessolution of N-Boc-N′-(L-phenylaninephenylaninyl) ehhylenediamine (3.9eq.) in methanol was added. Reaction allowed to proceed for 48 h. wasprecipitated in water, filtered and washed with water. The product wasfreeze-dried.

Procedure: In a round-bottom flask fitted with a stirrer compound (1eq.) was suspended in 1 ml of methanol. 4M HCl solution in dioxane wasadded (9 eq.). Reaction allowed to proceed for 2 hours. Mixture wasprecipitated in diethyl ether, washed and redissolved and treated withNaOH (till pH 10). Precipitate was filtered, washed with water andfreeze-dried.

1H NMR (DMSO-d6): 2.47 (dd, 1H), 2.8-3.1 (m, 7H), 4.49 (dt, 1H), 4.74(ddd, 1H), 7.08-7.31 (m, 10H), 7.81 (t, 1H), 8.2 (t, 1H), 8.23 (s, 1H),8.77 (d, 1H).

1.2. St-PBLG Polymer Synthesis

Briefly, γ-Benzyl L-glutamate N-carboxyanhydrides (0.5 g, 1.9 mmol) wasadded to a Schlenk tube fitted with a stirrer bar, a stopper and purgedwith 3 cycles of vacuum/Ar, and dissolved in 5 mL of the freshlypurified solvent. The 3-arm initiator was added and the mixture was leftstirring at 4° C. for 3 days under inert atmosphere. Finally, thereaction mixture was poured into a large excess of cold diethyl etherleading to a white polymer after isolation.

Yield: 70-90%. ¹H-NMR (300 MHz, DMF, δ) 8.58 (s, xH), 7.42 (s, 5H), 5.19(s, 2H), 4.21 (s, 1H), 2.81 (s, 2H), 2.45 (s, 2H). ¹³C-NMR (300 MHz,DMF, δ) 175.94 (s), 172.26 (s), 162.77-162.18 (m), 161.98 (s), 136.76(s), 128.87-127.75 (m), 66.05 (s), 57.13 (s), 35.41-34.17 (m), 32.48(s), 30.84, 30.30-29.04 (m), 27.28 (s), 25.99 (s). x: DP obtained/3arms.

TABLE 1 Variety of initiators used in the polymerization processes anddifferent DPs obtained for St-PBLGs, demonstrating the versatility ofthe technique. Mn^(a) Mn^(b) Star R₁ DP_(theo) (kDa) (kDa) DP^(a) DP^(b)D SE1 Ethyl 100 21.3 21.0  97  96 1.26 SE2

150 24.0 27.6 110 126 1.22 SE3 250 50.3 51.5 229 235 1.09 SH1 Hexyl  7516.4 n.d.  75 n.d. 1.25 SH2

150 23.9 23.6 109 108 1.23 SH3 250 51.5 52.7 235 240 1.17 SD1  75 15.716.9  72  77 1.13 SD2 DOOA 100 22.2 24.1 101 110 1.23 SD3

150 33.2 31.1 152 142 1.10 SD4 200 40.4 41.6 185 190 1.12 SS1 Cysteamine200 43.1 n.d. 196 n.d. 1.22

SP1 Phe-Ethyl 400 79.8 79.8 375 375 1.12

SPP1 Phe-Phe-Ethyl 150 31.5 28.9 144 132 1.06

^(a)Determined by NMR. ^(b)Determined by GPC. Mn and DP refer to numberaverage molar mass and degree of polymerization respectively. n.d. = notdetermined.

FIG. 1 shows routine characterization by GPC and CD of compounds offormula (I).

Following the general procedure for the polymerization described insection 1.2 for St-PBLG, and using the ethyl based initiator, a seriesof homopolypeptides and block or random copolypeptides have beensynthesized using the respective amino acid N-carboxyanhydride;epsilon-trifluoroacetate-L-Lysine N-carboxyanhydride, Benzyl-L-SerineN-carboxyanhydride, Sarcosine NCA. For random copolymers NCAs were mixedin the reaction environment prior to the addition of the initiator. Forblock copolymer, gamma-Benzyl L-glutamate N-carboxyanhydride was firstpolymerized through the addition of initiator followed by the additionof the second monomer once the first monomer was consumed.

TABLE 2 Variety of star-shaped polymers (compounds of formula (I))synthesized with other NCA monomers. Mn^(a) Mn^(b) Star R₁ DP_(theo)(kDa) (kDa) DP^(a) DP^(b) D St-PTLL 60 13.2 10.7 59 48 1.13 St-PTLL 25 5.0  3.6 22 16 1.03 St-PBLS

60 n.d.  9.7 n.d. 55 n.d. 25  4.0  3.4 23 20 1.10 St-PSAR 60  5.0  4.370 61 1.18 *St-PTLL: Star-Poly(epsilon-trifluoroacetate-L-Lysine),St-PBLS: Star-Poly(Benzyl-L-Serine), St-PSAR: St-Poly(Sarcosine)^(a)Obtained by GPC measurement in DMF/LiBr (0.1%). ^(b)Obtained by1H-NMR in deuterated TFA. n.d.: not determined.

TABLE 3 Star-shaped polymers (compounds of formula (I)) copolymers withBLG and SAR. DP_(t) DP_(t) Mn^(a) Mn^(b) DP^(b) DP^(b) Star R₁ _(BLG)_(SAR) (kDa) (kDa) _(BLG) _(SAR) D St-P(BLG- Ethyl 30 30 7.9 6.4 23 201.06 co-LSAR)

St-P(BLG- 30 30 8.5 6.6 24 20 1.05 b-LSAR) *St-P(BLG-co-SAR):St-poly(gamma-benzyl-L-glutamate-co-sarcosine) random copolymer,St-P(BLG-b-SAR): St-poly(gamma-benzyl-L-glutamate-block-L-sarcosine)block copolymer. ^(a)Obtained by GPC measurement in DMF/LiBr (0.1%).^(b)Obtained by ¹H-NMR in deuterated TFA.

FIGS. 2 and 3 show routine characterization by GPC of compounds offormula (I).

1.3. Deprotection of St-PBLG:

Different methods were followed depending on the initiator used: acidconditions were applied when ethyl and hexyl based initiators were used(initiators including ethyl or hexyl spacers). On the other hand, basicconditions were applied for DOOA (3,6-dioxa-8-octaneamine), andcysteamine based initiators synthesis (inititiators including DOOA orcysteamine spacers). Briefly:

a) Deprotection of St-PBLG with HBr in Trifluoroacetic Acid (TFA): in around bottom flask fitted with a glass stopper and a stirrer bar, 50 mgof St-PBLG (0.23 mmol, 1 eq. Glutamic Acid Unit, GAU) were dissolved intrifluoroacetic acid (TFA). Once dissolved, 2 eq. of HBr (48% v/v) percarboxyl group were added drop wise, and the mixture was stirred forfive hours. For big scale deprotection of St-PBLG, 16 hours were appliedfor full deprotection. Then, the solution was poured into a large excessof cold diethyl ether leading to a white solid after isolation.

b) Deprotection of St-PBLG in basic conditions: in a round bottom flask,40 mg of benzyl protected St-PBLG (0.183 mmol, 1 eq. GAU) was dissolvedin 16 mL of THF. The solution was cooled down to 4° C. and 2 eq. of NaOHper carboxylic group of the polypeptide block (14.7 mg, 0.369 mmol) wereadded in 2 mL of ddH₂O. The mixture was stirred for 16 hours. Finally,THF was removed and the residue was diluted with ddH₂O, concentrated andpurified by ultrafiltration (Vivaspin®, Molecular Weight Cut-offMWCO=3000 Da) or by size exclusion columns (G25).

Yields: 75-86%. ¹H-NMR (300 MHz, D₂O, δ) 8.2 (s, xH), 4.31-4.26 (m, 1H),2.38-2.14 (m, 2H) 2.10-1.80 (m, 2H) 2.10-1.80 (m, 2H). x: DP obtained/3arms. Depending on the initiator used, the corresponding signals of theethyl, hexyl, or DOOA were also present.

The deprotection of St-PBLG leads to the compound of formula (I)denominated St-PGA:

FIG. 4 shows routine characterization by ¹H-NMR and circular dichroismof compounds of formula (I)

Following the deprotection method a, the polypeptides synthesized intable 3 were deprotected and FIG. 5 shows the respective ¹H NMR in D₂O.

Example 2. Synthesis of Compounds of Formula (II)

In a one neck round bottom flask fitted with a stir bar and a stopper,200 mg of St-PGA (1.55 mol GAU, 1 eq.) were suspended in 10 mL of ddH₂O.Afterwards the eq. for the desired modification of4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl morpholinium (DMTMM)chloride (DMTMM-Cl) were added dissolved in 5 mL of ddH₂O (i.e. 128.7mg, 0.465 mmol, 0.3 eq. for 30% modification). After 10 minutes (0.93mmol 0.6 eq. for 30% modification) of the corresponding amine were addedand the pH was adjusted to 8 by adding some drops of 1 M NaHCO₃solution. Reaction was allowed to proceed overnight stirring at r.t.After this, as all by products were soluble in acid aqueous solution,either acid/base precipitation, dialysis (Vivaspin® MWCO 3000 Da), orsize exclusion chromatography with Sephadex G25 columns, was done inorder to purify the copolymer. A colorless amorphous solid was obtainedafter freeze-drying.

Yields: 80-90%. ¹H-NMR δ_(H) (300 MHz, D₂O) and chemical structures:

a) Star Poly(glutamic acid-co-propargyl glutamate), St-Prop(x): ¹H-NMRδ_(H) (300 MHz, D₂O): 8.2 (1H/(3DP),s), 4.30-4.02 (1H, m), 3.81 (2×H,s), 2.48 (1×H, s), 2.35-2.02 (2H, m), 2.01-1.65 (2H, m). x: molarpercentage of modification.b) Star Poly(glutamic acid-co-EG(n)N₃ glutamate), St-EG(2)N₃(x): ¹H-NMRδ_(H) (300 MHz, D₂O): 8.2 (1H/(3DP), 4.28-4.07 (1H, m), 3.65-3.51 (xH,m), 3.48 (2×H, t), 3.40-3.30 (2×H, m), 3.25 (2×H, d), 2.29-2.00 (2H, m),1.98-1.65 (2H, m). *R: 8 for EG2, 20 for EG6, 32 for EG9. x: molarpercentage of modification.c) Star Poly(glutamic acid-co-pyridyl cysteamine), St-PD(x): ¹H-NMRδ_(H) (300 MHz, D₂O): 8.4 (xH+1H/(3DP), m), 7.84 (2×H, m), 7.28 (xH, m),4.33 (1H, m), 3.48 (2×H, m), 2.95 (2×H, m), 2.3-1.9 (4H, m). x: molarpercentage of modification. d) St-Poly(glutamicacid-co-amino-ethyl-maleimide glutamate), St-Malei(X): ¹H-NMR δ_(H) (300MHz D₂O): 8.32 (1H/(3DP), s), 6.91 (2×H, s), 4.39 (1H, s), 3.80-3.16(4×H, m), 2.18 (4H, m). x: molar percentage of modification.e) St-Poly(glutamic acid-co-hydrazide-boc glutamate), St-hyd-boc(x):¹H-NMR δ_(H) (300 MHz, D₂O): 8.34-8.25 (1H/(3DP), s), 4.33 (1H, s),2.53-1.75 (4H, m), 1.45 (×1H, s). x: molar percentage of modification.f) St-Poly(glutamic acid-co-acetaldehyde) St-acetal(x): ¹H-NMR δ_(H)(300 MHz, D₂O): 8.29 (1H/(3DP), s), 4.32 (1H, s), 3.87-3.45 (×4H, m),3.18 (×1H, m), 3.02 (×1H, m), 2.45-1.85 (4H, m), 1.77-1.47 (×4H, m),1.19 (×6H, m). x: molar percentage of modification.

The synthesis were also carried out in organic solvents such as DMF,using the BF₄ salt of the DMTMM derivative.

FIG. 6 shows selected ¹H-NMR as part of routine characterization ofcompounds of formula (II).

Example 3. Study of Self and Co-Assembly Behavior Through BTA Motifs ofCompounds of Formula (I) and (II)

3.1. Self-Assembly of Compounds of Formula (I).

a) Physico-Chemical Evidences.

When further characterization of our star-shaped systems was carriedout, interesting data was found regarding compounds size. Small AngleNuclear Scattering (SANS) experiments have been performed as routinetechnique in the lab in order to elucidate size and solutionconformation of the compounds of the invention. When these architectureswere analyzed by SANS and after adequate data treatment and fittings(FIG. 7), gyration radius were found in the range of 70-160 nm, muchhigher than the ones expected for single St-PGAs (between 5-10 nm).These experiments were carried out at relatively high concentration (10mg·mL⁻¹) and therefore, self-assembly could be triggered. SANS fittinganalysis correlated these structures with “hard spheres with branchespointing outside”.

Moreover, when DLS measurements in PBS buffer pH 7.4 were performed, itwas found out that those systems undergo a concentration dependentself-assembly process. At low concentrations “unimers” of 5-10 nmdiameter size were identified, whereas bigger structures of around100-200 nm diameter size were formed at high concentrations. Thisphenomenon occurred in all star-shaped systems independently on thespacer in BTA core from the initiator (ethyl, hexyl or DOOA, cystamine,Phe-Ethyl). Nevertheless it did not occur in linear PGA (FIG. 8). Withincreasing concentrations, it could be clearly observed thedisappearance of the small structures and progressive appearance of thebigger ones, up to a point where only big structures of 100-200 nm size(diameter) were observed (2 mg·mL⁻¹). By plotting the scatteredintensity, Mean Count Rate (MCR) in Kcps obtained against concentration,a value of critical aggregation concentration (CAC) can be obtained withthe intersection of the two lineal curves (FIG. 8). This CAC value notonly represented the concentration above which aggregation processeswere taking place, but also represented the maximum concentration offree non-aggregated polymer species present in the sample under thatspecific conditions (temperature, ionic strength, pH).

Table 4 summarizes CAC values, hydrodynamic radius (R_(h)) and gyrationradius (R_(g)) obtained by DLS and SANS respectively, for severalSt-PGAs with different initiators and chain lengths. Similar R_(h)values were obtained for all of the measured stars, however, higher CACvalues were observed with greater chain lengths, with the exception ofthe DOOA initiator. This could be due to the hydrophilicity of thisspacer.

TABLE 4 Summary of CAC values, hydrodynamic radius (R_(h)) and gyrationradius (R_(g)) obtained by DLS and SANS for different star polymers. GAUR_(g) ^(d) Star R₁ arm^(a) CAC.^(b) R_(h) ^(c) (nm) (nm) SD2 Dooa 340.30 53.0 69.1 SD4 Dooa 62 0.30 47.7 91.1 SH3 Hexyl 78 0.40 62.7 80.8SE1 Ethyl 33 0.20 60.1 160.7 SE3 Ethyl 77 0.55 123.7 84.5 ^(a)GPC inDMF/LiBr at 8 mg · mL⁻¹. ^(b)Critical Aggregation Concentration (CAC)measured by DLS (mean count rate vs. concentration) in PBS at 20° C.^(c)DLS data at 2 mg · mL⁻¹ in PBS buffer pH 7.4 at 20° C. expressed byintensity mean. ^(d)SANS data (ILL, Grenoble, measured at 10 mg · mL⁻¹in PBS buffer pH 7.4 at 20° C.

Accordingly, a self-assembly process is proposed for these systems tolead bigger structures with hard sphere shapes bearing branching pointsoutside directed (FIG. 9). It must be noticed, that self-assemblyprocess of these St-PGAs represents a reversible and dynamic equilibriumbetween free non-aggregated species and large assemblies with broad sizedistributions. (FIG. 9.)

In order to unravel the molecular organization of BTA core within theassemblies, SANS contrast experiments were performed with D-labeled BTAarchitectures in LOQ SANS instrument at ISIS (UK). For that purpose, aDeuterium (D) labeled 3-arm initiator was synthesized in two steps using1,3,5-benzene tricarboxilic acid and N-Boc ethylendiamine both fullydeuterated (FIG. 10).

This system was studied through SANS contrast experiments both in H₂Oand D₂O solvents. Qualitatively, the contrast experiment in H₂O showed aprominent bump compared to the sample in D₂O. Aggregation of theself-assembling BTA core resulted in differences on the scatteringlength density between the hydrophobic domain and the polymer backboneexpressed in our system as a “bump” in I(Q) versus Q plot at high Qvalues (FIG. 11). This feature provided a direct indication of acharacteristic ‘short’ dimension in the structure, suggesting thepresence of BTA self-assembled domains. This pointed out the presence ofBTA core domains in contrast to a random distribution of BTA moietiesalong the nanostructure, in agreement with previous reports inliterature. This fact confirms that BTA central core is the drivingmotif for the assembly of these architectures rather than simply thestar shape.

When observed under the microscope using Cryo-Transmission ElectronMicroscopy (TEM), the star-shaped polymer bearing BTA motifs at thecore, exhibited homogenous globular shaped nanoparticles of about 80-100nm diameter with relatively low dispersities, further confirming thefindings obtained in the first SANS experiment and DLS analysis (FIG.12).

b) Stimuli-Responsiveness.

With size-concentration dependence verified, the effect of differentstimuli such as temperature and ionic strength were further investigatedusing St-PGA without any modification as a model system. Size dependenceon ionic strength of media was investigated after the first evidencesfound (see Table 5) when measuring the same sample in ddH₂O or PBSbuffer 0.1 M pH 7.4.

TABLE 5 Size determination of St-PGA (ethyl based initiated) by DLS (PBSand ddH₂O) and DOSY NMR. Compound R_(h) ^(a) (nm) R_(h) ^(b) (nm) R_(h)^(c) (nm) R_(h) ^(d) (nm) R_(h) ^(e) (nm) St-PGA 36.4 69.1 123.7 2.7124.4 *Data obtained of a 2 mg · mL⁻¹ sample from ^(a)DOSY NMR in D₂O.^(b)DLS number mean in ddH₂O. ^(c)DLS intensity mean in ddH₂O. ^(d)DLSnumber mean in PBS 7.4. ^(e)DLS intensity mean in PBS 7.4.

It can be concluded that presence of salt, and therefore, modulation ofthe ionic strength, highly affects the self-assembly equilibrium byshifting it towards unimer region. In the absence of salts, no unimerscould be observed by DLS. Thereafter, ionic strength was further studiedas we decided to investigate the influence of different salts onaggregate size (FIG. 13). Sodium chloride (NaCl), guanidiniumhydrochloride (GuHCl), and sodium Sulphate (Na₂SO₄) were chosen due totheir different nature. As can be observed in FIG. 13, the scatteredintensity is progressively reduced with increasing salt content, beingNa₂SO₄ the most disruptive. Furthermore, R_(h) (mean number) dependenceon salt content reveals disassembly of aggregates just by addition of 50mM of any salt.

Size dependence on concentration was studied at 37° C. in theconcentration range of 1 to 10 mg·mL⁻¹ St-PGA. As it can be observed inFIG. 14 a sudden increase in size was observed above 5 mg·mL⁻¹ from ˜70nm to ˜100 nm. Size dependence on temperature was also found when thesystem was studied by DLS measurements at 10 mg·mL⁻¹ in the temperaturerange between 10 and 60° C.

3.2. Self-Assembly of Compounds of Formula (II).

For that purpose, several star polymers based on ethyl-BTA initiatorwere modified with alkynes and azides using the optimizedpost-polymerization techniques either both in the same polymer chain orin different polymers to yield compounds of formula (II). From 5 to 50%of GAUs (glutamic acid units) of St-PGAs were modified withpropargylamine and NH₂EG(2)N₃ respectively. One polymer was also duallymodified with 10% alkyne and 20% azide mol GAUs. Those polymers whereanalyzed by DLS and CAC was calculated (FIG. 15). As negative controlfor the study, linear alkyne modified PGAs (5 and 10 mol % GAUs) werealso measured leading to absence of aggregation processes in theconcentration range studied

TABLE 6 Summary of CAC values and hydrodynamic radius (R_(h)) obtainedby DLS of compounds of formula (II). Mod. R_(h) ^(d) R_(h) ^(e) CompoundGAU arm^(a) GAU^(b) CAC^(c) (nm) (nm) St-prop(5) 50 5 0.60 44.0 67.2St-prop(10) 50 10 0.50 38.5 77.3 St-prop(20) 50 20 0.40 37.4 68.6St-prop(30) 50 30 0.35 49.2 95.5 St-prop(50) 50 50 0.35 45.1 90.6St-EG(2)N₃(5) 50 5 0.50 2.3 69.0 St-EG(2)N₃(10) 50 10 0.55 2.7 58.2St-EG(2)N₃(20) 50 20 n.d.* 2.6 75.2 St-EG(2)N₃(30) 50 30 n.d.* 2.5 65.8St-EG(2)N₃(50) 50 50 n.d.* 2.6 71.1 n.d. = not determined. *A CAC couldnot be calculated in the concentrations range employed. Aggregation (ifoccurs) might be found over 2 mg · mL⁻¹. ^(a)GPC in DMF/LiBr at 8 mg ·mL⁻¹. ^(b)Data obtained by ¹H-NMR in mol %. ^(c)CAC measured by DLS inPBS at 20° C. and size measured by DLS at 2 mg · mL⁻¹ in PBS at 20° C.by ^(d)Number mean, and ^(e)Intensity mean.

Assemblies' morphology was also investigated through Cryo-TEM. As shownin FIG. 16, this morphology does not vary significantly from the parentcompound with the different chemical modifications introduced. In allcases globular aggregates in the range of 100 nm were found. In generalthese results are in good agreement with those found for the parentcompound and also with DLS and SANS data obtained for these series ofcompounds.

In a second example, in order to validate the versatility of thisapproach, different polymer chain modifications (introduction of thiols,maleimides, hydrazides and acetals) in order to perform other covalentcapture strategies were implemented. First of all, the synthesis ofstar-shaped polymers bearing activated di-thiol units (usingpyridyl-cysteamine, PD), star polymers with maleimide groups (usingNH₂—CH₂CH₂-maleimide, malei), stars bearing hydrazide (hyd) groups andacetals was performed. Compound identity was determined by ¹H-NMR, (FIG.6) Their aggregation behavior was also studied by DLS as for theprevious compounds, leading to aggregated structures of around 100 nmupon increasing the concentration.

TABLE 7 Summary of CAC values and hydrodynamic radius (R_(h)) obtainedby DLS of compounds of formula (II). R_(h) ^(d) R_(h) ^(e) Compound GAUarm^(a) Mod. GAU^(b) CAC^(c) (nm) (nm) St-PD(4 50 4 0.6 141.55 194.4St-PD(10) 50 10 0.55 168.78 144 St-PD(21) 50 21 0.5 121.88 142.5St-PD(35) 50 30 0.45 84.68 n.d. St-PD(44) 50 44 0.3 n.d. 138.9 St-PD(60)50 60 n.d.* n.d. 88.3 St-malei(5) 50 5 0.40 82.3 38.5 St-malei(10) 50 100.35 74.6 31.9 St-malei(35) 50 35 0.30 n.d. n.d. St-hyd(5) 50 5 0.5 74.632.9 St-hyd(10) 50 10 0.4 81.6 35.8 n.d. = not determined; *C.A.C. couldnot be calculated in the concentrations range employed. Aggregation (ifoccurs) might be found over 2 mg · mL⁻¹. ^(a)GPC in DMF/LiBr at 8 mg ·mL⁻¹. ^(b)Data obtained by ¹H-NMR in mol %. ^(c)CAC measured by DLS inddH₂O at 20° C. and size measured by DLS at 2 mg · mL⁻¹ ddH₂O at 20° C.by ^(d)Number mean. ^(e)DOSY data in D₂O.

3.3. Co-Assembly of Compounds of Formula (II).

Studies to assess co-assembly where done using DLS, by observation ofCAC value shift of one of the compounds upon addition of constant amount(always below its CAC) of the second component. Two different series ofsolutions were prepared for the CAC determination experiments:St-EG(2)N₃(5), St-prop(10), and the same series but with addition of thesecond component in a concentration below their CAC. FIG. 17 shows theplots of scattered intensity against variable concentration of one ofthe components keeping constant the concentration of the counterpart(always bellow their CAC). It can be seen a decrease in CAC value inboth cases when the second compound was added to the solution. Thesefindings somehow suggest a synergy in the formation of mixed assembliesthrough co-assembly processes what is in good agreement with previousreports on PEG modified BTA species but also block-copolymer systems.

Pulsed-gradient spin-echo NMR spectroscopy, known as diffusion NMRspectroscopy (or DOSY NMR), allows determining the self-diffusioncoefficient of the species present in solution. Then, co-assemblyprocess was tested by using a sample containing St-prop(10) above itsCAC (2 mg·mL⁻¹) in the presence of St-EG(2)N₃(5) below its CAC (0.1mg·mL⁻¹). As it can be seen in FIG. 18, ¹H NMR spectra showed thesignals corresponding to each of the components employed in DOSY NMRanalysis. After data treatment, it can be seen that compound St-prop(10)shows the characteristic diffusion coefficient of self-assembled species(5.03·10⁻¹² m²·s⁻¹) expected for the concentration studied. However,compound St-EG(2)N₃, that, at 0.1 mg·mL⁻¹ should present a largerdiffusion coefficient when compared to the self-assembled constructs,reduced its diffusion coefficient in one order of magnitude from(3.12·10⁻¹¹ m²·s⁻¹) to (5.24·10⁻¹² m²·s⁻¹), being virtually equivalentto that found for St-prop(10) component. These results suggest thatalthough St-EG(2)N₃ is below the CAC, it moves along with theself-assembled constructs from the counterpart St-prop(10), and thus,indirectly confirms that these architectures are able to co-assemble.

Moreover, confirmation of co-assembly process was assessed with the helpof NOESY experiments. As observed in FIG. 19, a clear NOE correlationwas found for propargyl and ethylene glycol signals, a result thatconfirms the spatial proximity between both groups.

Example 4: Synthesis of Compounds of Formula (III)

A general scheme of the proposed methodology to self-assemble thestar-shaped polyglutamates into well-defined morphologies and stabilizethe aggregates through covalent cross-linking is depicted in FIG. 20including all the chemical and structural variations. The following nonlimiting example is intended to illustrate the bottom up approach (seeFIG. 21) employed for the construction of these complex carriersemploying the propargyl and azide functionalized derivatives.

Methodology for copper catalyzed alkyne-azide coupling (CuAAC) of St-PGAderivatives: using the compounds of formula (II), star-shaped polymersSt-prop(10) (Star PGA based on BTA-ethyl and modified with propargylamine units 10 mol %) and St-EG(2)N₃(5) (Star PGA based on BTA-ethyl andmodified with oligoethylene glycol azide units 5 mol %): Those polymerswere chosen in order to have an excess of propargyl units to ensurecomplete conversion, as the reaction will be performed in equimolarratio of both functionalities. The reaction was carried out in ddH₂O(constructs were present in aggregated state as seen before), using aconcentration to ensure the only presence of big structures within thepolymeric mixture (ratio 1:1, 2 mg·mL⁻¹). The mixture was firstlysonicated for 5 minutes in order to promote homogenization. Then, 5 eq.of sodium ascorbate in ddH₂O solution were added. Then, the mixture wasdegassed by performing two freeze-pump-thaw cycles. One eq. of CuSO₄ wasweighted under N₂ flow and added in ddH₂O solution to the reactionmixture. The final complete mixture, was degassed by performing anotherfreeze-pump-thaw cycle and left to react at 40° C. in an oil bathprotected from light. Complete conversion was achieved after 3 days,according to ¹H-NMR (triazole signal at 7.8 integrates for 5 mol %).Other coupling chemistries encompassing i) di-thiol Chemistry, ii)thiol-maleimide Chemistry, iii) hydrazine-aldehyde Chemistry(Wolff-Kishner) were also carried out.

The products obtained were characterized by ¹H-NMR and results shown inFIG. 22.

Additionally, the clicked system was studied by DLS measurements incomparison with a physical mixture 1:1 of both components separatelyafter sonication. Dilution experiments were performed by diluting bothsamples up to 32 fold 1 mg·mL⁻¹ stock solution. In the case of thephysical mixture, two different structures were already found at thefirst dilution (1:2 ratio). Nevertheless, for the clicked construct,only big structures of about ˜80-100 nm diameter were encountered, evenat 1/32 of the initial concentration (˜0.03 mg·mL⁻¹) (FIG. 23). Thesmall decrease in the assemblies found for the clicked system (from 45to 30 nm in radius) might be due to the low eq. of effectivecross-linking groups (in this case azide, 5 mol %) resulting in anincomplete cross-linking of the self-assembled nanostructures.

Cryo-TEM pictures of the clicked system confirmed the formation ofspherical structures with a diameter size˜100 nm (FIG. 24).

Covalent capture using di-thiol chemistry, was performed atconcentrations of 10 mg mL⁻¹ for each compound in ddH₂O for di-thiolchemistry and PBS buffer at pH 7.4 for thiol-ene, due to the need ofcontrolling the pH over reaction time in order to guarantee maleimidegroup stability. After purification by dialysis, the success of theentrapment was ratified by ¹H-NMR. (FIG. 22) In the case of di-thiolchemistry confirmation was achieved by disappearance of the aromaticsignals corresponding to pyridyl groups while CH₂ signals of cysteaminewere kept, in the case of di-thiol chemistry. For thiol-ene reactions,the absence of the characteristic maleimide peak around 6.7 as well asthe pyridyl signals were indicatives of effective couplings. DLS and TEMmeasurements confirmed the covalent capture leading to stable structuresof around 100 nm diameter.

Example 5. Synthesis of Conjugates Comprising Compounds of Formula (I),(II) or (III) and an Agent

Modification of glutamate residues is carried out under analogousconditions for all the compounds comprised in formulas I, II and III. Tosimplify, the general synthetic strategy is illustrated for a generalpoly-L-glutamic acid backbone as depicted bellow:

5.1. Conjugation of Oregon Green Cadaverine to Compounds of Formula (I),(II) or (III):

For macromolecular therapeutics and nano-sized drug delivery systems,fluorescent labeling is commonly applied to allow intracellulartrafficking studies, conjugate cell-specific localization and/or in vivofate and PK. Probes such as the fluorophore Oregon Green (OG) have beenextensively reviewed for cellular studies to determine cell uptake andbinding. To this aim, the conjugated probe must fulfill somerequirements such as high stability of the probe itself as well asstability of the linkage to ensure adequate carrier monitoring. On theother hand, a minimal percentage of probe loading is desirable in orderto avoid data misinterpretation due to changes in polymer conformationresulting from changes in charge and solubility. In order to fulfill allthat criteria, less than 1 mol % of OG was conjugated through anon-biodegradable amine bond. Conjugation of OG to the clicked systemwas carried out either by using diisopropylcarbodimide(DIC)/Hydroxybenzotriazole (HOBt) as carboxylic acid activators inorganic solvents or DMTMM.Cl in aqueous solution. The protocol of OGconjugation was previously established and routinely used with DIC/HOBtin Dr Vicent laboratory, ensuring 80-90% conjugation efficiency of thefluorescence dye. A schematic representation of polymers labeling isdepicted in the following scheme:

In a round two necked bottom flask fitted with a stirrer bar and twoseptums, 29 mg of compounds of formula (I), (II) or (III) (0.225 mmolglutamic acid units or GAU, 1 eq.) was weighed and dissolved in 1.5 mLof dry DMF under N₂ flow. Of N, N′Diisopropylcarbodiimide (1.12 μL) andDIC (0.85 mg, 0.00674 mmol, d=0.806 g/mL, 0.03 eq.) were added and thereaction was left to proceed for 5 min at room temperature. Afterwards,Hydroxybenzotriazole, HOBt (1 mg, 0.00674 mmol, 0.03 eq.) was addeddirectly. The reaction was then left to proceed for 10 min before OregonGreen Cadaverine (1 mg, 2.25·10⁻³ mmol, 0.01 eq.) was added. The pH wasadjusted to 8 by adding ˜100 μL of DIEA. The mixture was left stirringovernight protected from light. Finally, the solvent was removed undervacuo at room temperature and the product was dissolved in 300 μL ofwater and then adding ˜50 μL of NaHCO₃ 1M. The solution was purified bySephadex PD10 column eluting with distilled water. The Oregon Green (OG)loading was calculated by fluorescence using a Victor²Wallace™ platereader with excitation filter of 490 and emission filter of 535. Acalibration curve with OG was first performed. Yield: 95%. OG loading:0.8 mol glutamic acid unit.

5.2. Conjugation of Cy5.5 to Compounds of Formula (I), (II) or (III).

Briefly, in a one-neck round bottom flask, PGA-based polymer wasdissolved in ddH₂O (1 eq. GAU). Then, the carboxylic groups wereactivated using DMTMM.Cl (i.e. 0.02 eq. for 2% modification). Reactionwas allowed to proceed for 10 minutes. After that time, Cy5.5 (i.e. 0.02eq. for 2% modification) was added in ddH₂O. The pH was adjusted to 8 byadding sodium bicarbonate 1 M. Reaction was left to proceed for 24hours, protected from light. For purification, the products weresubmitted to both Sephadex G25 and dialysis using Vivaspin® MWCO 5000.

Cy5.5 content estimation was carried out by fluorescence (A_(em): 595nm, A_(ex): 680 nm) after the building of an appropriate calibrationcurve of Cy5.5 dye in PBS buffer.

Yields: 60-70%. Conjugation efficiency 70-90%.

5.3. Conjugation of DO₃AtBu-NH₂ to Compounds of Formula (I), (II) or(III):

As for OG conjugation, DIC/HOBt as carboxylic acid activators in organicsolvents or DMTMM.Cl in aqueous solution were used.

In a two-neck round bottom flask fitted with a stir bar and two septums,300 mg (of St-PGA), 110 GAU, 2.32 mmol GAU, 1 eq.) was dissolved in 20mL of anhydrous DMF under nitrogen flow. Then, 53 μL of DIC (88 mg, 0.70mmol, 0.3 eq.) were added and the reaction was left to proceed for 5 minat room temperature. Afterwards, HOBt (94 mg, 0.70 mmol, 0.3 eq.) wasadded directly. The reaction was then left to proceed for 10 min beforeDO₃AtBu-NH₂ (282 mg, 0.46 mmol, 0.2 eq.) was added for 20% modification.The pH was adjusted to 8 by adding ˜100 μL of DIEA. The mixture was leftstirring for 48 hours at room temperature and protected from light.Finally, the solvent was partially removed under vacuo, precipitatedinto a large excess of cold acetone, filtered off and washed three timeswith cold acetone. A pale yellow solid was obtained after drying. Thepercentage of modified GAU was calculated as 20% mol GAU, according tothe tBu groups signal at 1.4 ppm in comparison with the alpha proton ofPGA backbones in ¹H-NMR spectra. Conjugation efficiency: 100%. Yield:75%.

Deprotection of DO₃A tBu-NH:

Two different protocols were used depending on the compound nature. Forconstructs without any sensitive group to trifluoroacetic acid (TFA)conditions, the first protocol was applied. The use of Triisopropylsilylether (TIPS) in the second protocol was introduced in order to preventdisulfide bonds breakage during deprotection conditions.

Protocol 1. The construct was dissolved in CH₂Cl₂/TFA (3/2, v/v) mixtureand left under vigorous stirring for 16 hours at r.t. After that time,the solution was precipitated by pouring into a large excess of colddiethyl ether. Pale yellow solid was obtained after filtering, washingwith diethyl ether and drying under vacuum. Complete deprotection wasachieved as confirmed by ¹H-NMR. Yields: 80-90%.

Protocol 2. The construct was dissolved in TFA/H₂O/TIPS (95/2.5/2.5,v/v) mixture and left stirring at r.t. during 3 hours. After that, thecontents were precipitated into a large excess of cold diethyl ether. Apale yellow solid was collected, washed with diethyl ether and driedover vacuum. Complete deprotection was confirmed by ¹H-NMR analysis.Yields: 80-90%

5.4. Radiolabelling with ¹¹¹In of Compounds of Formula (I), (II) or(III).

As example, St-PGA-DO₃A-¹¹¹In was prepared by dissolving 51.3 mg ofSt-PGA-DO₃A in deionized water to a final concentration of 10 mg/mL.Then, 0.25-0.5 mL of this dissolution was transferred into a microwavetube and the pH was adjusted to 3.5-4 by adding HEPES buffer and HCl 2M. Next 7-27 MBq of ¹¹¹InCl₃ in HCl 0.05 M was added and the reactionmixture heated at 90° C. for 5 min by using a laboratory microwave withmonomodal radiation (Discover Benchmate, CEM). After that, the reactionmixture was cooled down with nitrogen gas. The reaction was stoppedafter 5 min at room temperature by the addition of 50 μL of 50 mMethylenediaminetetraacetate acid (EDTA). St-PGA-DO3A-¹¹¹In was purifiedfrom unreacted ¹¹¹In-EDTA by exclusion molecular chromatographycartridge (Bio Gel P-6, BioRad) using phosphate buffered saline (pH=7)as eluent, at flow rate 0.5 mL/min. The elution profile was determinedby fractionating, 0.77 mL per fraction, and measuring each with a dosecalibrator (VDC 405, Veenstra). Radiochemical yield (RY) was calculatedas percentage of the activity in each fraction eluted from the molecularexclusion cartridge of the total activity purified and corrected for thedecay.

5.5. DO3A-Gd³⁺ Labeling for MRI of Compounds of Formula (I), (II) or(III).

In a one-neck round bottom flask, the corresponding DO3A bearing polymeras sodium salt form (1 eq. of modified DO3A GAU units) was dissolved inPBS 0.1 M pH 7.4. Then, GdCl₃ (1 eq.) dissolved in ddH₂O was droppedinto the main solution. During this process, pH was monitored andremained constant to 8. The degree of Gd (III) complexation wasdetermined by titrating aliquots during reaction process using4-(2-pyridylazo) resorcinol which turns from yellow to orange in thepresence of free Gd). No free Gd was detected after 5 hours reactiontime. The reaction was then stopped and purified by dialysis usingVivaspin® MWCO 5000. Absence of free Gd was again confirmed by using thetitrating method described before with the dialyzed contents.

Example 6: Validation of Compounds of Formula (I) as Carriers

6.1. Degradation with Cathepsin B:

To be sure that the enzyme-dependent biodegradability of thepolyglutamate-based stars had not been compromised by the architecture,all polymers were incubated in presence of the lysosomal enzymecathepsin B. St-PGA was degraded in presence of the lysosomal enzymecathepsin B as its linear counterpart. To test the profile and ratio ofthe degradation of the polymers by cathepsin B, solutions of the St-PGAs(3 mg/mL) were prepared. 3 mg exactly were weighed and 700 μL of acetatebuffer 20 mM, pH 6, 100 μL of EDTA 2 mM, 100 μL of DTT 5 mM were added.Finally, 6.25 units of Cathepsin B (100 μL of a solution of 25 units ofcathepsin B in 400 μL of acetate buffer pH 6 20 mM were added. CathepsinB needs pH 6 to be active, the DTT solution is added to activateCathepsin B and the solution of EDTA was added to complex the freecations (mainly Ca²⁺ that inactivates cathepsins). Once the solutionswere prepared, aliquots of 100 μL were picked at different time points(t=0, 0.5, 1, 2, 4, 8, 24, 48 and 72 hours) after homogenization of thesolutions. Meanwhile, the samples were kept at 37° C. under stirring.The aliquots taken were frozen and later analyzed by GPC. To evaluatethe mass of the conjugates, 100 μL of 3 mg/mL conjugate solution in PBSwas injected in the GPC using two TSK Gel columns in series G2500 PWXLand G3000 PWXL with a Viscoteck TDA™ 302 triple detector 87 with UVdetection coupled. The mobile phase used was PBS 0.1 M, flow 1 mL/min

As expected, the star polymers were found to be degraded by thelysosomal enzyme with similar kinetics independently of the MW andinitiator used for the polymerization (FIG. 25a ).

6.2. Cell Viability.

Another key feature for the validation of the compounds of the inventionas potential drug delivery carriers or imaging probes is their toxicityin cell cultures. To this respect, 72 hours MTS assays(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumcolorimetric agent assay) were performed against SHSY5Y human derivedneuroblastoma cell line as well as in HUVEC (human umbilical veinendothelial cells). Polymers were found to be non-toxic up to 3 mg/mL(FIGS. 25b and c ).

6.3. Cellular Uptake.

Understanding of cellular internalization mechanisms used bynanopharmaceuticals has become a key player in the field of drugdelivery. Nanomedicines mainly use endocytic vesicles or endosomes,which in turn employ a complex mechanism to address the differentmolecules to specific intracellular locations. It can be said thatcharge, shape, material composition, and surface functional groups arebasic physico-chemical parameters that determine cell entry ofnanomedicines by endocytic pathway.

Confocal microscopy techniques and flow cytometry are routinely usedwith fluorescence-labeled polymers in order to evaluate their uptake bycells. Live-cell confocal imaging, allows visualizing traffickingbetween multiple compartments within individual living cells over time,avoiding any possible artifacts derived from fixation protocols. On theother hand, flow cytometry give us semi-quantitative information aboutthe mechanism of internalization.

Flow cytometry (cell uptake and binding) together with live-cellconfocal microscopy analysis (subcellular fate and pathway) in SHSY5Yhuman derived neuroblastoma cell line, were used to study cellulartrafficking of the OG-labeled star-shaped polymers (FIG. 26). Flowcytometry experiments were carried out at different temperatures, 37° C.(to measure the total uptake) and 4° C. (to measure cell binding) inorder to determine the presence of energy dependent or non-dependentinternalization mechanisms, such as endocytosis or diffusion,respectively. It is worth mentioning that all experiments were done inpresence of the cathepsin B inhibitor CA-047 in order to avoid thedegradation of the polyglutamic acid chains along the incubationperiods.

Live-cell confocal imaging allows visualizing trafficking betweenmultiple compartments within individual living cells over time, avoidingany possible artifacts derived from fixation protocols. Results fromboth techniques clearly showed an energy-dependent mechanism ofinternalization due to the absence of uptake at 4° C. as observed byflow cytometry. This was further confirmed with the confocal microscopystudies at 2 hours post-treatment with an OG labeled polymer in SHSY5Ycells following a pulse-chase experiment, where co-localization in thelysosomes was observed upon the use of lysosomal marker Lysotracker Red(FIG. 26b ).

Interestingly, the St-PGA-OG showed a significant increase in celluptake at 5 hours when compared with linear-PGA-OG conjugate of similarMW (FIG. 27). This might be attributed to the inherent propertiesassigned to the star-shaped polymers. As general basis, star polymershave a more compact structure, presumably with globular shape, and havelarge surface areas, increased concentrations of functional end groupsfor polymers with equal molecular weight, and unique rheologicalproperties which make them optimal platforms for drug delivery andimaging among other biological applications.

6.4. Biodistribution and Pharmacokinetics (PK).

To further validate the synthesized nanocarriers, in vivobiodistribution as well as pharmacokinetic profiles (PK) were obtainedby radioactivity measurements. For that purpose, a gamma emittingradionuclides ¹¹¹In was introduced into the star-shaped PGAs throughcomplexation chemistry as previously explained. In order to accomplish astable complexation of the metal radioisotope, the incorporation ofbifunctional chelating agents into the polymer backbone is required. Themost commonly used chelating agents for ¹¹¹In are based on polyaminecarboxylic acids such as diethylene triamine pentaacetic acid (DTPA),1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or1,4,7-triazacyclododecane-1,4,7-tetraacetic acid (NOTA). For thebiodistribution of radiolabeled PGA based architectures, DOTA derivativechelating agent with a free amine group suitable for conjugation(DO₃A-NH₂) was selected. It is well-known that, DO₃A-NH₂ forms stablecomplexes with several M²⁺ and M³⁺ ions such as ⁶⁸Ga or ¹¹¹In.Therefore, 20% mol GAU of DO₃A-tBu-NH₂ was effectively conjugated viaamide bond to a St-PGA₁₁₀ (D 1.23).

Conjugation efficiency was almost quantitative (since 10% mol GAU waspursued) with a reasonable mass yield of 75%. The percentage ofmodification was calculated according to ¹H-NMR analysis by comparingthe corresponding integral of the CH alpha of PGA (4.24 ppm) with the 27protons of the tBu groups at 1.40 ppm (FIG. 28). The tBu groups werethen easily deprotected using the mixture CH₂Cl₂/TFA (3/2, v/v).Finally, the St-PGA-DO₃A polymer was labelled with ¹¹¹In as describedpreviously. Radiochemical yields were ≥85% for St-PGA-DO₃A-¹¹¹In in allsynthesis (as shown in FIG. 29).

Animal experiments to test the biodistribution and PK profile were thencarried out with i.v. injected doses between 37 KBq and 2.5 MBq of¹¹¹In-labelled polymers (1-20 μg/g body weight) and monitored up to 24hours (4-5 mice were sacrificed per time point 0.5, 1, 2, 4, 8 and 24hours). Blood and organs were extracted and radioactivity was measuredex vivo in the gamma counter.

According to the results obtained from the biodistribution, where thehigher percentage of injected dose (ID) corresponded to the kidneys, itcan be concluded that these new polypeptide architectures follow renalexcretion profiles with no specific accumulation in any organ (FIG. 30).

The biodistribution profile obtained for St-PGA was then compared withthe one obtained for its linear counterpart of similar MW (100 GAU, D:1.20). The biodistribution of linear PGA was previously performed using⁶⁸Ga radioisotope, therefore, only short times (up to 3 hours) could berecorded due to radionuclide decay (about 68 min for ⁶⁸Ga). If shorttime points (0.5 h, 1 h and 2 h) of the % ID/g tissue of PGA-DO₃A-⁶⁸Gaand St-DO₃A-¹¹¹In are compared, a general greater accumulation in allthe organs of star shaped polymer is observed, in comparison with theone found in the linear PGA construct as shown in FIG. 31.

Although the plasmatic profiles are similar for both compounds,differences can be drawn when we compared the PK parameters obtained forPGA-DO₃A-⁶⁸Ga with St-PGA-DO₃A-¹¹¹In. The linear polymer was notdetected after 4 hours post administration. Their biological or terminalhalf-life estimated resulted to be 13 times higher for the star polymer,this fact could be in part attributed to the use of differentradionuclides for the study. The use of ¹¹¹In allowed to study andestimate the PK parameters of the stars providing results more reliabledue to the higher semidesintegration period for ¹¹¹In (2.1 days)compared to ⁶⁸Ga (68 min).

TABLE 8 Main St-PGA-[¹¹¹In]-DO3A and PGA-[⁶⁸Ga]-DO3A pharmacokineticparameters estimated by a 2-compartment model following the equationC(t) = Axe^((−ALPHAxt)) + Bxe^((−BETAxt)). Values represent Mean ± SD.Parameter St-PGA[¹¹¹In]-DO3A- PGA-[⁶⁸Ga]-DO3A- A (% ID/mL) 22.33 ± 5.13 35.00 ± 12.88 B (% ID/mL) 0.04 ± 0.01 4.35 ± 2.78 ALPHA (h⁻¹) 4.73 ±0.42 7.28 ± 2.56 BETA (h⁻¹) 0.06 ± 0.04 1.18 ± 0.59 AUC (% ID · h/mL)5.45 ± 0.76 8.50 ± 0.67 t_(1/2) ALPHA (h) 0.15 ± 0.01 0.10 ± 0.03t_(1/2) BETA (h) 12.05 ± 7.96  0.59 ± 0.29 Cl (mL/h) 18.35 ± 2.55  11.77± 0.93  Vss (mL) 46.34 ± 44.44 5.25 ± 2.52

In the case of the two compartment model a number of volume terms can bedefined. V_(ss) is the appropriate volume of distribution (Vd) whenplasma concentrations are measured in steady state conditions. ThisV_(ss) value is about 9 times higher for the star polymer compared tothe linear one, meaning a greater distribution of the carrier. TheClearance value (Cl) from the central compartment is slightly higheralso in the star-shaped polymer (18.35 vs 11.77 mL/h for linear PGA).The renal clearance value of inulin (a model compound that is excretedonly by glomerular filtration and is not subject to tubular secretion orre-absorption) has being established to be around 20 mL/h by in FVBmice. This value is really close to the value obtained for the starpolymer. Thus it could be claimed that the polymer is cleared out onlyby glomerular filtration. In the case of linear PGA, the value isslightly smaller. This could be explained by the binding of the compoundto plasmatic proteins, reducing the glomerular filtration, or if thelinear polymer could be reabsorbed in the tubules.

Example 7: Validation of Compounds of Formula (III) as Carriers

7.1. Cell Viability.

Cell viability against SHSY5Y cell line of the three chemicallydifferent clicked architectures was studied. All of them resultednon-toxic up to 3 mg·mL⁻¹ when tested at 72 hours of incubationfollowing an MTS protocol for cell viability determination. Results areshown in FIG. 32.

7.2. Cellular Uptake.

Flow cytometry (cell uptake and binding) together with live-cellconfocal microscopy analysis (subcellular fate and pathway) in SHSY5Yhuman derived neuroblastoma cell line, were used to study cellulartrafficking of the OG-labeled clicked stars (compounds of formula III).Uptake experiments were carried out at different temperatures, 37° C.(total uptake) and 4° C. (binding) in order to determine the presence ofenergy dependent or non-dependent internalization mechanisms, such asendocytosis or diffusion, respectively. It is worth mentioning that allexperiments were done in the presence of cathepsin B inhibitor CA-047 inorder to avoid possible degradation of PGA chains along the incubationperiods. Results were represented by means of cell associatedfluorescence (CAF) over incubation time. CAF represents the percentageof positive cells multiplied by fluorescence intensity and divided by100, always removing CAF of control cells (without treatments) in orderto avoid any artifacts from autofluorescence phenomena.

Results shown in FIG. 33 demonstrate the energy-dependent mechanisms ofinternalization (endocytosis) in due to absence of uptake at 4° C. asobserved by flow cytometry. As it could be expected, this globularshaped structure was fast internalized, showing around 95% of positivecells already at 15 minutes (FIG. 33a ). Furthermore, when thisconstruct was compared with the linear PGA and non-clicked star PGA, asignificant increase in cell-associated fluorescence (CAF) was observed(FIG. 33b ). Not only such compound goes through a faster uptake(according to both CAF and % positive cells) but also, the amount ofconstruct internalized is significantly greater when compared with theother 2 systems. Furthermore, co-localization with the lysosomal markerLysotracker Red was found in confocal microscopy images (FIG. 34).

Example 8. Validation of Compounds of Formula (III) as Carrier to Crossthe Blood Brain Barrier (BBB)

8.1. Synthetic Strategy and Characterization.

In order to validate the systems as adequate carriers for intravenousadministration, biodistribution experiments were done. The clicked starswere labeled with DO3A-Gd³⁺ for MRI techniques and Cy5.5 forfluorescence optical imaging techniques. Furthermore, in order to ratifythe versatility of the systems to be used as carriers through the BBB,the targeting ligand ANGIOPEP-2 (ANG-2) currently in Phase II clinicaltrials was linked to the polymers. Synthetic route of these conjugatescan be seen in FIG. 35.

Briefly, DMTMM-Cl was employed in order to activate the carboxylic acidsto allow the introduction of DO3AtBu-NH₂ in the first place, followed byCy5.5 in the synthesis of the non-targeted system. DO3A modified unitswere quantified by ¹H-NMR. On the other hand, Cy5.5 content estimationwas carried out by fluorescence (prior calibration curve of Cy5.5 dye inPBS buffer was obtained.

For the non-targeted construct, tBu protecting groups from DO3A wereeasily removed at this point, using the mixture TFA:TIPS:H₂O(95:2.5:2.5). In the case of the targeted polymer, cysteamine-2TP unitswere introduced again by post-polymerization modification in aqueousmedia prior to the introduction of Cy5.5. Quantification was determinedas 10 mol % of GAU by ¹H-NMR. Then, the tBu protecting groups from DO3Awere removed, and ANG was conjugated following previous strategies bymeans of disulfide bonding. Finally, Gd³⁺ was complexed to DO3A bearingconstructs using a 1:1 eq. (DO3A:GdCl₃) ratio. The reaction took placein PBS 0.1 M at pH 8 (GdCl₃ precipitation was observed at lower pHs) andmonitored by titration using 4-(2-pyridylazo)resorcinol. This titratingagent turns from yellow to orange in the presence of free Gd³⁺. After 5hours reaction time, no free Gd³⁺ was detected. The reaction waspurified by dialysis and absence of free Gd³⁺ was again tested.Conjugates physico-chemical characteristics are summarized in Table 9.

TABLE 9 Conjugate physico-chemical characteristics for in vivobiodistribution by fluorescence. mol % GAU wt % mol % GAU/ mol % GAU/Compound wt % DO3A Gd wt % Cy5.5 wt % ANG St-Click-DO3A-Gd- 10.0 mol %12.0 0.5 mol % — Cy5.5 20.3 wt %  3.1 wt %  St-Click-DO3A-Gd-   10 mol %10.4 0.5 mol % 1.5 mol % Cy5.5-ANG 17.6 wt %  2.7 wt %  13.8 wt % 

The Z-potential of the clicked architectures before, and after surfacemodifications was recorded in ddH₂O at 20° C. and the results aredepicted in FIG. 36. As it can be observed, surface modifications withDO3A-tBu and cysteamine-2TP, significantly decrease the negativeZ-potential obtained for the clicked structure with all the carboxylicgroups unmodified and presumably exposed at the surface.

The introduction of the negatively charged Cy5.5 within the structureresulted in an increase on Z-potential obtained. Finally, when ANG-2peptidic sequences where conjugated, Z-potential dramatically decreaseto almost neutral, probably due to a shielding effect provided by the 19aa sequences.

Furthermore, size of the systems was estimated by TEM to be in the rangeof 70-100 nm diameter (FIG. 37)

8.2. In Vivo Evaluation of Compounds of Formula (III) as Carriers toCross the BBB.

Biodistribution experiments were carried out using C₅₇Bl/6 mice andfluorescence techniques taking profit from Cy5.5 dye on the polymericcarriers. Targeted and non-targeted architectures were administered i.v.through the tail vein to isofluorane anesthetized mice, at a dose of4.15 mg·Kg⁻¹ Cy5.5 eq. Two animals were then sacrificed at differenttime points (1, 3, 7, 14 and 24 hours). Prior to sacrificed, mice werefirst anesthetized with a lethal anesthesia cocktail, blood wasextracted from the cava vein, and perfusion with saline was carried outin order to accurately determine the amount of compound in the brain.Then, organs were extracted and their fluorescence was measured usingthe red filter in MAESTRO™. For fluorescence quantification, normalizeddata was obtained by taking always the same pixel area for all organsexpressed as average signal (counts·s⁻¹). A calibration curve of thecompounds in the same MAESTRO™ was carried out in order to estimate thefluorescence corresponding to the injected dose. Biodistribution dataobtained from non-targeted and targeted polymer is depicted in FIGS. 38and 39.

When both compounds were compared, no major differences inbiodistribution were encountered as it can be observed in FIG. 39. Renalexcretion profiles could be observed in both cases. However, thetargeted compound was found to accumulate in a higher extend in organssuch as liver and kidney. Notably, when the biodistribution data fromthese bigger architectures was compared with that from the non-clickedstars, a greater accumulation in the lungs at early time points wasobserved. This fact was in good agreement with the nature of thearchitectures used, since sizes above 100 nm tend to accumulate inlungs. Hence, this family of architectures could have a potential use inorder to target lung diseases such as lung cancer. Nevertheless, thesecarriers also demonstrated to be safe as not weight loss in the animalswas observed. Besides, lung accumulation was significantly diminishedover time, validating them as possible carriers.

Important to note, the ANG bearing compound offered greater brainaccumulation at early time points when compared to the non-targetedcounterpart. Nonetheless, similar accumulation was found for bothcompounds at late time points such as 24 hours. Remarkably, the amountfound in the brain in both cases was between 1-1.5% ID, which is 20-30times greater than the one obtained for non-clicked stars (0.05% ID). Asmentioned before and according to literature, the normal % ID for thosesystems which are able to reach the brain is usually between 1-2% ID,with the maximum obtained with 4%.

Example 9. Validation as Carrier to Treat Neurodegenerative Disease

9.1. Synthetic Protocol and Characterization.

We aimed to obtain combination conjugates for systemic administrationwith synergistic effect using the neuroprotective-neurorescuer propargylmoieties and the neuro-antiinflamatory curcuminoids, looking for a newtherapeutic strategy in AD.

Briefly, in a two-necked round bottom flask, fitted with a stirrer barand two septums, the corresponding star polymer was dissolved in 10 mLof anh. DMF under nitrogen atmosphere. After that, 1.5 eq. of DMTMM·BF₄of the desired percentage of GAU modification was added in 5 mL more ofanh. DMF. Reaction was left to proceed for 10 minutes. Then, 1.5 eq. ofthe desired percentage of GAU modification of bisdemethoxycurcumin(BDMC) were added to the reaction mixture, followed by a catalyticamount of DMAP. The pH was checked to be around 7. Reaction was thenleft to proceed for 72 hours. For purification, the mixture was pouredinto a large excess of diethyl ether. After isolation, the yellowishsolid was converted into sodium salt form by careful addition of NaHCO₃1 M. Then, the aqueous solution was washed with diethyl ether till noyellowish coloration was found in the organic phase. Finally, theproduct in aqueous phase was purified by dialysis using Vivaspin® MWCO5000, and freeze-dried. BDMC contain was determined by UV-VIS at 415using a calibration curve with free BDMC. FDC was estimated by HPLCfollowing the method: eluent A was ddH₂O and eluent B was acetonitrile.Samples were analyzed using the following gradient: from 40% B to 80% Bover 20 min using Lichrospher 100 RP 18, 5.0 μm (dimension:length×ID)=125×4.0 mm). BDMC retention time (tr) 5.98 minutes.Experiments were done per triplicate. % of free drug was established byperforming a calibration curve with BDMC dissolved in the mixtureddH₂O/Acetonitrile (50/50) and injected under the same HPLC conditions.

TABLE 10 Physico-chemical characteristics of BDMC-conjugates throughbottom-up approach, conjugates of formula (II) and (III). TDC wt % FDCwt % of TDC Conjugate (Abs 415 nm) (Abs 415 nm, HPLC) St-BDMC 0.5 <1St-BDMC 1 <1 St-BDMC 2.5 <1 St-EN(2)N3(5)-BDMC 1.25 <1 St-Click-BDMC2.00 <1 St-Click-BDMC 4.00 <1 *TDC: total drug content; FDC: free drugcontent.9.2. Cell Viability.

Firstly, cytotoxicity of BDMC bearing polymers was explored up to 15 μMdrug-eq. According to previous studies found in literature, acurcuminoid concentration range of 0.1-1 μM should be enough to induce atherapeutic benefit by diminishing oxidative stress. Moreover, the IC50value for Aβ aggregation and lipid peroxidation of curcuminoids is alsofound in that concentration rage, indicating that such a dose should beenough in order to produce antioxidant and anti-inflammatory effects. Asit can be observed from FIG. 40, non-significant toxicities up to 10 μMdrug-eq. were found. The compound St-Click-BDMC with 4 wt % of BDMC wasselected for further investigations (100% cell viability at 10 μM).

9.3. Drug Release Profiles.

Since a pH degradable linker (ester) was used for the conjugation ofBDMC, the kinetics of drug release under hydrolytic conditions wasconsequently studied. Samples of St-Click-BDMC 4 wt %, (selected fromcell viability experiments) were incubated at 37° C. at different pHsincluding 5.0 (lysosome), 6.5 (endosome) and 7.4 (blood) up to 96 hours.A sustained and controlled drug release profile was obtained after HPLCanalysis. About 20% of the conjugated drug was released within 2 days atpH 5.0 whereas pH 6.5 and 7.4 showed a much slower release profile (seeFIG. 41).

9.4. Prevention of Fibril Formation In Vitro.

In order to achieve proof of concept, activity of the compounds waschecked in a first attempt using an accepted model based on the use ofHen Egg White Lysozyme (HEWL) for protein amyloid formation. HEWL is amonomeric protein composed of 129 amino acids with helix richconformation, and it represents one of the best known model proteins tostudy protein aggregation. It has been demonstrated that under acidic pHthis protein undergo amyloid aggregation (FIG. 42). Hence, activity ofseveral BDMC bearing conjugates, as inhibitors of fibril formation waschecked by Thioflavin T (ThT) fluorescence measuring, which is incorrelation with fibril formation. ThT is a benzothiazole salt used as adye to visualize and quantify the presence or fibrillization ofmisfolded protein aggregates, or amyloid, both in vitro and in vivo(i.e. plaques composed of amyloid beta found in the brains ofAlzheimer's disease patients). ThT Assay measures changes offluorescence intensity of ThT upon binding to amyloid fibrils (FIG. 42).The enhanced fluorescence can be observed by fluorescence microscopy orby fluorescent spectroscopy. The spectroscopic assay is normally used tomonitor fibrillization over time.

Then, several BDMC bearing compounds and free BDMC, for comparison, attwo different concentrations (10 and 50 Mm BDMC-eq.) were incubated for24 hours with HEWL (2 mg·mL⁻¹ solution) at 60° C. under vigorousmagnetic stirring, and at low pH in order to favor amyloid aggregation.PBS solutions and the polymeric carrier were used as positive controls.It is worth mentioning that, no fibrillation was found neither when HEWLwas incubated at r.t. nor when no magnetic stirring was used. Aliquotsof the fibril samples were taken at different time points and mixed withThT aliquots for 5 minutes. Finally, fluorescence was measured in aVictor™ Wallace (λ_(exc) 450 nm and A_(em) 510 nm) and backgroundfluorescence from curcuminoid subtracted (FIG. 43). By this assay, itcould be concluded that the polymer conjugates exhibits a fibrilinhibitor behavior slightly better (although no significantly different)than free BDMC. It was also clear that, activity of the conjugates wasmainly due to the presence of curcuminoid and not to the PGA chains. Theuse of higher concentrations (50 μM drug-eq.) did not improve theresults obtained when compared with lower concentrations (10 μMdrug-eq.). 10 μM BDMC-eq was selected then, as the concentration to moveforward. These results were further confirmed by TEM, as it can beobserved in FIG. 44.

9.5. Effects of St-Click-BDMC on Aβ Induced Neurotoxicity in HippocampalOrganotypic Cultures.

The neuroprotective effect of the curcuminoid bearing polymericstructure was evaluated in organotypic cultures from entorhinalcortex-hippocampus. In order to study neuroprotection, the experimentaldesign involved pretreatments with the conjugate prior to an Amyloid-βpeptide (Aβ₁₋₄₂) triggered injury. This ex vivo model has beenpreviously validated to determine neurotoxicity and constitutes aneffective manner to identify the neuroprotective effect of moleculeswith real therapeutic potential against AD. The organotypic cultures ofslices containing both entorhinal cortex and hippocampus are anexcellent ex vivo model to monitor the structure and physiology of theseregions of the limbic system. They preserve the principal circuits ofhippocampus, including its main excitatory input coming from theentorhinal cortex. Besides, they can be maintained for long periods oftime, optimal to evaluate pharmacological activity on neurons or glialcells of the different treatments upon time. Hippocampus and entorhinalcortex are among the most affected regions in AD, accumulating a highdensity of extracellular deposits of Aβ peptide, and are partiallyresponsible of the progressive memory loss and cognitive impairmentobserved in this neurological disorders.

Previous work has provided strong evidence that the synthetic peptideAβ₁₋₄₂ is able to induce neural injury in this type of organotypicculture. Hence, the aim was to analyze this cell damage and its putativeprevention by a pretreatment with the St-Click-BDMC 4 wt % usingpropidium iodide (PI) staining (FIGS. 45 and 46). PI is a polar compoundimpermeable to intact cell membranes, but capable to penetrate damagedcells and to bind to nuclear DNA, providing a bright red fluorescence.This labeling, allow us the quantification of the density of degeneratedcells in a given region. In our case, the region of interest (ROI) wasthe CA1 region of hippocampus (cornus ammonis 1), where several studieshave found neurodegenerative effects induced by Aβ peptides.

Viability of the organotypic cultures in the presence of St-Click-BDMCand absence of Aβ peptides was firstly investigated (48 hoursincubation). Slices were stained with PI, fixed and finally analyzed byconfocal microscopy. Our polymer conjugate, up to 0.2 μM BDMC-eq, didnot induce significant changes in IP positive nuclei density whencompared to control cultures (0.005 μM F_((4, 18))=11.096, ρ=1; 0.05 μMF_((4, 18))=11.096, ρ=1; 0.2 μM F_((4, 18))=11.096, ρ=0.41). At 0.5 μMdrug-eq. concentration an increase in cell death was observed(F_((4, 18))=11.096, p<0.0001) (See FIG. 45).

The concentrations of 0.05 and 0.2 μM drug-eq. were then selected inorder to have the maximum tolerated concentration to provideneuroprotective effects in Aβ₁₋₄₂ treated cultures. In this case,organotypic slices were pretreated with the polymer conjugate 48 hoursbefore Aβ cell death induction. Thereafter, cultured slices were treatedwith a second dose of conjugate and Aβ₁₋₄₂ (1 μM final concentration).48 hours later, cell death was quantified after staining with PI,fixation and analysis by confocal microscopy. In this case, pretreatmentwith 0.2 μM BDMC-eq. induced a significant increase in cell death(F_((5,9))=9.574, ρ=0.006) but not in the case of 0.05 μM BDMC-eq.Cultures treated with Aβ₁₋₄₂ increased cell death when compared tocontrols (vehicle (F_((5,19))=9.574, ρ=0.0001), and 0.05 μM drug-eq. ofpolymer conjugate (F_((5,19))=9.574, ρ=0.006)) as shown in FIG. 46.Pretreatment of cultures with 0.05 μM polymer conjugate(F_((5,19))=9.574, ρ=0.005) or 0.2 μM (F_((5,19))=9.574, ρ=0.026) beforeAβ₁₋₄₂ addition induced a significant decrease in the density of PIlabeled nuclei when compared with cultures treated only with Aβ₁₋₄₂peptide. (FIG. 46).

Overall, the construct bearing BDMC tested in organotypic cultures showsno toxicity after 48 hours of treatment at the different concentrationstested, except for 0.5 μM concentration. When repeated doses wereapplied (in the case of the pretreatment experiment), the 0.2 μMconcentration resulted toxic for the non Aβ₁₋₄₂ peptide treatedcultures, however, this concentration was effective for Aβ₁₋₄₂ toxicityprevention. Pretreatment with polymer conjugate at either 0.05 or 0.2 μMof drug-eq. significantly reduced cell death in Aβ₁₋₄₂ peptide treatedcultures. As 0.05 μM concentration resulted enough to producesignificant neuroprotective effects against Aβ₁₋₄₂ neurotoxicity withoutbeing toxic, this concentration was selected to move forwards. Furtherexperiments are ongoing in order to identify the possible mechanisms ofneuroprotection followed by our constructs.

9.6. Preliminary Studies of St-Click-BDMC Demonstrating Safety on aGenetically Modified Alzheimer's Disease Model

For that purpose, the mouse strain ArcAbeta was used as Alzheimer'smouse in vivo model. As our idea is to tackle the disease from aneuroprotective point of view, young animals (from 8-11 months) werechosen. Since this mouse model starts to accumulate plaque burden ataround 6-9 months of age, excessive and irreversible amounts of Aβplaques will not be present. Firstly, in vivo safety is a go/no go stepfor any tested compound in order to proceed with its validation.Therefore, a pilot study with St-click-DO3A-Gd-Cy5.5.-BDMC was designedwith a dose schedule selected based on PK studies. In this firstexperiment, animal weight was monitored as a proof of safety uponsuccessive administrations of the compound. Three different groups ofanimals were chosen: wild type animals as control (×2), ArcAbeta animalsused as non-treated controls injected with saline (×7) and ArcAbetaanimals treated with the compound at a comparable dose as that used inthe biodistribution studies (2 mg·Kg⁻¹ BDMC eq.) (×7).

Animals were injected six times within two weeks without showing signsof toxicity as it is depicted in FIG. 47 where no weight loss of thetreated animals was observed.

Example 10. Validation as Carrier to Cancer Applications

10.1. Synthetic Protocol and Characterization.

We aimed to obtain conjugates for systemic administration looking for anew therapeutic strategy in cancer research. As a model drug,Doxorubicin (DOX) was conjugated via pH-labile bonds (hydrazone) tocompounds of formula I, II or III, cross-linked with 20% PD groups. Thesynthesis of compounds of based on compounds of formula (III) isdescribed in detail herein. First of all, hydrazone linker wasintroduced into the polymer backbone of compounds of formula III.Briefly, in a one-necked round bottom flask with a stir bar and twoseptums, 1 eq. of compound of formula III (acid form) was dissolved inthe required volume of anh. DMF (i.e. 40 mL for 400 mg). Afterwards theeq. for the desired modification of DMTMM·BF₄ were added, dissolved inanh. DMF (i.e. 0.9302 mmol, 0.3 eq. for 20% modification). The pH waschecked to be 5. After 10 min, the corresponding amine(tert-butyl-carbazate) was added, dissolved in t anh DMF. The pH wasadjusted to 7-8 with DIEA. The reaction was allowed to proceed for 48hours, stirring at r.t. under N₂ atmosphere. The product wasprecipitated in diethyl ether, filtered-off and dried. The Bocprotecting group was removed with TFA. The reaction was allowed toproceed for 45 minutes, stirring at room temperature. The product wasthen precipitated into cold diethyl ether, washed three times withdiethyl ether, and twice with milli-Q® water pH3. A colourless amorphoussolid was obtained after freeze-drying. FIG. 48 shows the proton NMRs ofexamples of compounds of formula (II and III) synthesized following thisprocedure. Next, % mol GAU of DOX was incorporated into the compound byreaction in DMSO, catalyzed with four drops of acetic acid, the reactionwas allowed to proceed for 48 hours, stirring at r.t., under N₂atmosphere. After evaporation of half of the volume, the polymers wereprecipitated in cold diethyl ether by first adding THF in order toimprove the miscibility of DMSO in ether. The precipitate was dissolvedin 6 mL of DMF and purified by size-exclusion chromatography on aSephadex® LH-20 column. The first fraction was isolated and the solventwas evaporated using a vacuum pump. The dried product was suspended inmilli-Q® water and converted into the sodium salt form by adding NaCO₃.The excess of salts was removed by size-exclusion chromatography on aSephadex® G-25 column. A colourless amorphous solid was obtained afterfreeze-drying. The total drug loading was measured by UV/VISspectroscopy at 480 nm resulting in 2.5 mol % Glutamic Acid Units (GAU)DOX (7.85 wt %). Yields: 56-85%

10.2. Cell Viability.

Mouse 4T1 breast cancer cells were cultured in RPMI 1640 (withL-Glutamine and 25 mM Hepes) medium and incubated at 37° C. with anatmosphere of 5% carbon dioxide. MTS cell viability assay was performedwith quantification after 72 hours.

In each well of a sterile 96-well microtiter plate, 2000 cells wereseeded and incubated for 24 hours under the same conditions mentionedbefore. A stock solution of 1 mg/mL (DOX eq.) of the compound of theinvention was prepared in PBS and diluted with medium to reach a finalconcentration of 0.1-50 μg/mL. The medium from six wells was removed andreplaced by 100 μL of each dilution. After 72 hours of incubation, 10 μLof MTS/PMS (20:1) was added to each well. The cells were then incubatedfor 3 more hours.

The absorbance at 570 nm was measured spectrophotometrically usingVictor²Wallace™ plate reader. For calculations, the absorbance oftreated cells was compared with the absorbance of untreated controlcells, representing 100% cell viability.

As is represented in FIG. 49, the compound exerted a toxicity similar ofthat of free DOX, and therefore, it represents a promising candidate.

10.3. Drug Release.

DOX release profile under different pH conditions was explored. Thecompound of the invention was dissolved in different pH PBS solutions(pH 5 and 7.4) at a concentration of 3 mg/mL. Then, 2 mL of thissolution was added in a Float-A-lyzer G2 device (MWCO 1000 Da). Thedevice floated under stirring in 100 mL of the corresponding buffersolution. All samples were incubated in an oven at 37° C. during theexperiment. Aliquots of 1000 μL PBS solution wherein the device floatedwere taken at different time-points (0 min, 15 min, 30 min, 45 min, 1 h,2 h, 4 h, 8 h, 12 h, 24 h, 32 h, 48 h, 56 h, 72 h, 80 h, 96 h, 168 h)and replaced with, 1000 μL of fresh PBS solution was added to maintainthe volume of 100 mL. After freeze-drying, each aliquot was dissolved in120 μL DMSO 10% and 100 μL of this was added on a 96-well dark plate.The concentration of DOX on the plates was measured by fluorescencespectroscopy by triplicate. A calibration curve of DOX in DMSO 10% wasprepared from dilutions by following the same procedure. Thefluorescence (excit 450/emi 595) nm was measured using Victor²Wallace™plate reader.

pH-dependence drug release was obtained since there was almost norelease at pH 7.4 and about 18% drug release at pH 5.0 at 80 hours. FIG.50.

The invention claimed is:
 1. A compound of formula (I) below, comprisinghomopolypeptides or random or block co-polypeptides:

or its salts, solvates or isomers, wherein: m, m′, m″, n, n′, n″, o, o′and o″ are integers independently selected from 0 to 500, wherein atleast one of them is ≥1; R₆ to R₈, R_(6′) to R_(8′) and R_(6″) to R_(8″)are independently selected from H and methyl; I₁ to I₃ are independentlyselected from the group consisting of H; halogen; Deuterium; and(C₁-C₂₀)-alkyl; R₂ to R₄, R_(2′) to R_(4′) and R_(2″) to R_(4″) areindependently selected from the group consisting of:

X₁ and X₂ are independently selected from the group consisting of H; N;NH₂; and Z; X₃ and X₄ are independently selected from the groupconsisting of H; and Z; y and y′ are integers between 0 and 3; andy+y′=2 or 3; y″ and y′″ are integers between 0 and 2; and y+y′=1 or 2 Zis selected from the group consisting of H; metallic counterion;inorganic counterion; and an amino acid protecting group; R₁, R_(1′) andR_(1″) are radicals independently selected from the group consisting of:

A₁, A₂, A₃ and A₄ are radicals independently selected from the group asdefined for R₂ to R₄, R_(2′) to R_(4′), and R_(2″) to R_(4″); L₁ is aradical independently selected from the group consisting of a(C₁-C₅₀₀)-alkyl, wherein one or more H is optionally substituted by: (1)(C₃-C₃₀)-cycloalkyl, (2) a C-radical derived from a ring system with 1-6rings, each ring being independently saturated, partially unsaturated oraromatic, the rings being isolated or fused and having 3-20 members eachmember independently selected from the group consisting of C, CH, CH₂,CO, N and NH, (3) OH, (4) NR_(a)R_(b), (5) ONR_(c)R_(d), (6) CN, (7)halide, (8) SH₂, (9) SR_(e)R_(f), (10) N(H)NH₂, (11) R_(g)COR_(h), (12)COOR_(i), (13) CON(R_(j))(R_(k)), (14) R_(l)N(R_(m))CON(R_(n))₂, (15)(C₁-C₃₀)-alkene, (16) (C₁-C₃₀)-alkyne, (17) N₃, (18)R_(o)CH(OR_(p))(OR_(q)), (19) R_(r)CH(SR_(s))(SR_(t)), (20)R_(n)Boron(OR_(v))(OR_(w)), (21) COR_(x); and wherein one of more C areindependently replaced by (C₃-C₃₀)-cycloalkyl, aryl,aryl-(C₁-C₃₀)-alkyl, NR_(y)R_(z), CO, O, S, Boron, halide, P and(O—CH₂—CH₂)_(B); B is an integer between 1 and 500; R_(a), R_(b), R_(c),R_(d), R_(e), R_(f), R_(h), R_(i), R_(j), R_(k), R_(m), R_(n), R_(p),R_(q), R_(s), R_(t), R_(v), R_(w), R_(x), R_(y) and R_(z) are radicalsindependently selected from the group consisting of H; (C₁-C₃₀)-alkyl;(C₁-C₃₀)-alkylphenyl; phenyl (C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl,wherein one or more carbons are optionally substituted by an heteroatomselected from the group consisting of O; S; F; N; NH; P; and CO; R_(g),R_(l), R_(o), R_(r) and R_(u) are radicals independently selected fromthe group consisting of (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl; phenyl;(C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl, wherein one or more carbons areoptionally substituted by an heteroatom selected from the groupconsisting of O; S; F; N; NH; P; and CO; R₅, R_(5′) and R_(5″) areradicals independently selected from the group consisting of H; and(C₁-C₅₀₀)-alkyl, optionally substituted by: (1) (C₃-C₃₀)-cycloalkyl, (2)a C-radical derived from a ring system with 1-6 rings, each ring beingindependently saturated, partially unsaturated or aromatic, the ringsbeing isolated or fused and having 3-20 members each memberindependently selected from the group consisting of C, CH, CH₂, CO, Nand NH, (3) OH, (4) NR_(a)R_(b), (5) ONR_(c)R_(d), (6) CN, (7) halide,(8) SH₂, (9) SR_(e)R_(f), (10) N(H)NH₂, (11) R_(g)COR_(h), (12)COOR_(i), (13) CON(R_(j))(R_(k)), (14) R_(l)N(R_(m))CON(R_(n))₂, (15)(C₁-C₃₀)-alkene, (16) (C₁-C₃₀)-alkyne, (17) N₃, (18)R_(o)CH(OR_(p))(OR_(q)), (19) R_(r)CH(SR_(s))(SR_(t)), (20)R_(u)Boron(OR_(v))(OR_(w)), (21) COR_(x); and wherein one of more C areindependently replaced by (C₃-C₃₀)-cycloalkyl, aryl,aryl-(C₁-C₃₀)-alkyl, NR_(y)R_(z), CO, O, S, Boron, halide, P and(O—CH₂—CH₂)_(B); B is an integer between 1 and 500; R_(a), R_(b), R_(c),R_(d), R_(e), R_(f), R_(h), R_(i), R_(j), R_(k), R_(m), R_(n), R_(p),R_(q), R_(s), R_(t), R_(v), R_(w), R_(x), R_(y) and R_(z) are radicalsindependently selected from the group consisting of H; (C₁-C₃₀)-alkyl;(C₁-C₃₀)-alkylphenyl; phenyl (C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl,wherein one or more carbons are optionally substituted by an heteroatomselected from the group consisting of O; S; F; N; NH; P; and CO; R_(g),R_(l), R_(o), R_(r) and R_(u) are radicals independently selected fromthe group consisting of (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl; phenyl;(C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl, wherein one or more carbons areoptionally substituted by an heteroatom selected from the groupconsisting of O; S; F; N; NH; P; and CO.
 2. The compound according toclaim 1, wherein: I₁, I₂ and I₃, are radicals independently selectedfrom the group consisting of H; Deuterium; and F; R₅, R_(5′) and R_(5′″)are identical between them and selected from the group consisting of H;CO—(C₁-C₂₀)-alkyl; CONH—(C₁-C₂₀)-alkyl; and pyroglutamate.
 3. Thecompound according to claim 1, wherein: R₂═R_(2′)═R_(2″),R₃═R_(3′)═R_(3″), and R₄═R_(4′)═R_(4″), and each of them isindependently selected from the group consisting of:

X₁ and X₂ are independently selected from the group consisting of H; N;—NH₂; and Z; y and y′ are integers between 0 and 3; and y+y′=2 or 3;R₁═R_(1′)═R_(1″) and R₁ is selected from:

A₁, A₂, A₃ and A₄ denote the side residues of hydrophobic amino acidsand they are selected from the group consisting of:

and combinations thereof.
 4. A compound of formula (II):

or its salts, solvates or isomers wherein: R₁ to R₈, I₁ to I₃, n, and o,are defined as in claim 1; m is an integer between 2-500; x is a numberfrom 0.01*m to 0.5*m; R_(2′″) is a radical selected from the groupconsisting of:

X₁ is H; y is 0 or 1; CL is a radical selected from the group consistingof a (C₁-C₅₀₀)-alkyl, wherein one or more H is optionally substitutedby: (1) (C₃-C₃₀)-cycloalkyl, (2) a C-radical derived from a ring systemwith 1-6 rings, each ring being independently saturated, partiallyunsaturated or aromatic, the rings being isolated or fused and having3-20 members each member independently selected from the groupconsisting of C, CH, CH₂, CO, N and NH, (3) OH, (4) NR_(a)R_(b), (5)ONR_(c)R_(d), (6) CN, (7) halide, (8) SH₂, (9) SR_(e)R_(f), (10)N(H)NH₂, (11) R_(g)COR_(h), (12) COOR_(i), (13) CON(R_(j))(R_(k)), (14)R_(l)N(R_(m))CON(R_(n))₂, (15) (C₁-C₃₀)-alkene, (16) (C₁-C₃₀)-alkyne,(17) N₃, (18) R_(o)CH(OR_(p))(OR_(q)), (19) R_(r)CH(SR_(s))(SR_(t)),(20) R_(u)Boron(OR_(v))(OR_(w)), (21) COR_(x); and wherein one of more Care independently replaced by (C₃-C₃₀)-cycloalkyl, aryl,aryl-(C₁-C₃₀)-alkyl, NR_(y)R_(z), CO, O, S, Boron, halide, P and(O—CH₂—CH₂)_(B); B is an integer between 1 and 500; R_(a), R_(b), R_(c),R_(d), R_(e), R_(f), R_(h), R_(i), R_(j), R_(k), R_(m), R_(n), R_(p),R_(q), R_(s), R_(t), R_(v), R_(w), R_(x), R_(y) and R_(z) are radicalsindependently selected from the group consisting of H; (C₁-C₃₀)-alkyl;(C₁-C₃₀)-alkylphenyl; phenyl (C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl,wherein one or more carbons are optionally substituted by an heteroatomselected from the group consisting of O; S; F; N; NH; P; and CO; R_(g),R_(l), R_(o), R_(r) and R_(u) are radicals independently selected fromthe group consisting of (C₁-C₃₀)-alkyl; (C₁-C₃₀)-alkylphenyl; phenyl;(C₁-C₃₀)-alkyl; and (C₃-C₈)-cycloalkyl, wherein one or more carbons areoptionally substituted by an heteroatom selected from the groupconsisting of O; S; F; N; NH; P; and CO.
 5. The compound according toclaim 4, wherein: I₁, I₂ and I₃, are radicals independently selectedfrom the group consisting of H; Deuterium; and F; R₅ is selected fromthe group consisting of H; CO—(C₁-C₂₀)-alkyl; CONH—(C₁-C₂₀)-alkyl; andpyroglutamate.
 6. The compound according to claim 4, wherein: each R₂,R₃, and R₄ is independently selected from the group consisting of:

X₁ and X₂ are defined as in claim 1; y and y′ are defined as in claim 1;R₁ is selected from the following groups:

A₁, A₂, A₃ and A₄ denote the side residues of hydrophobic amino acidsand they are selected from the following groups or combinations thereof:

L₁ is as defined in claim
 1. 7. The compound according to claim 4,wherein: R_(2′″) is selected from the group consisting of:

X₁ is H; y is 0 or 1; CL is selected from the group consisting of:

R₉ and R₁₁ to R₁₇ are independently selected from the group consistingof:

p and s are integers independently selected from 0 to 500; R₁₀ isselected from H and (C₁-C₄)-alkyl.
 8. A cross-linked self-assembled starpolymer comprising a recurring unit of formula (III):

or its salts, solvates or isomers wherein: R₁ to R₈, I₁ to I₃, m, n ando are defined as in claim 1; x is a, number from 0.01*m to 0.5*m;R_(2′″) is selected from the group consisting of:

X₁ and y are defined as in claim 4; CL₁ is defined as CL in claim
 4. 9.The cross-linked self-assembled star polymer according to claim 8,wherein: I₁, I₂, and I₃, are radicals independently selected from thegroup consisting of H; Deuterium; and F; R₅ is selected from the groupconsisting of H; CO—(C₁-C₂₀)-alkyl; CON(H)—(C₁-C₂₀)-alkyl; andpyroglutamate.
 10. The cross-linked self-assembled star polymeraccording to claim 9, wherein: each R₂, R₃, and R₄ is independentlyselected from the group consisting of:

X₁ and X₂ are defined as in claim 1; y and y′ are defined as in claim 1;R₁ is selected from the following groups:

A₁, A₂, A₃ and A₄ denote the side residues of hydrophobic amino acidsand they are selected from the following groups or combinations thereof:

L₁ is as defined in claim
 1. 11. The cross-linked self-assembled starpolymer according to claim 8, wherein: R_(2′″) is selected from thegroup consisting of:

X₁ and y are defined as in claim 4; CL₁ is selected from the groupconsisting of:

R₉ and R₁₁ to R₁₇ are selected from the group consisting of:

p and s are integers independently selected from 0 to 500; R₁₀ isselected from H and (C₁-C₄)-alkyl.
 12. A conjugate comprising thecompound of formula (I) as defined in claim 1, the compound of formula(II) as defined in claim 4, or the cross-linked self-assembled starpolymer of formula (III) as defined in claim 8, and at least an activeagent which is linked to the compound or the self-assembled starpolymer.
 13. The conjugate according to claim 12 wherein the at leastactive agent is selected from the group consisting of an activeingredient and an imaging agent, or combinations thereof.
 14. Theconjugate according to claim 13, wherein the at least active ingredientis selected from the group consisting of anticancer agent,antimetastatic agent, anti-inflammatory agent, antioxidant,antiapoptotic, proapoptotic, neuroprotective agent, immunostimulantagent, antioxidants, agent capable to trigger tissue repair and/orregeneration, anti-amyloidotic agent, and plaque/protein aggregatesdisrupting agents.
 15. The conjugate according to claim 14, wherein theat least active ingredient is selected from the group consisting of:vincristine, vinblastine, amiloride, chloroquine, blafiomycyn, fasudil,bisphosphonate, primaquine, meclofenamate, tonabersat, disulfiram,cyclophosphamide, paclitaxel, dendrotoxin, doxorubicine, methotrexate,epirubicine, dinaciclib, buparlisib, palbociclib, veliparib, megestrol,examestane, goserelin, tamoxifen, fulvestrant, trastuzumab, lapatinib,pertuzumab, selegiline, rasagiline, ladostigilM30, demethoxycurcumin,curcumin, and bisdemethoxycurcumin.
 16. The conjugate according to claim12, wherein the conjugate comprises an amount of the at least an activeagent in the range between 1 to 70% w/w based on the mass ratio of theat least active agent to the conjugate.
 17. A pharmaceutical, diagnosticor theranostic composition comprising at least one conjugate as definedin claim 12 together with one or more appropriate pharmaceutical ordiagnostically acceptable excipients.
 18. A method for treatment of aneurodegenerative disorder, neurological disease, cancer, infectiousdisease, disorder related to aging, neuro-inflammation, demyelinatingdisorder, multiple sclerosis, ischemic disorder, ischemia-reperfusioninduced damage, amyloydotic disease, cardiomyopathy, spinal cord injury,immune disorder, inflammatory disorders, rare disease, wound healing andlysosomal storage disease comprising administering the conjugateaccording to claim
 12. 19. A carrier comprising a compound of formula(I) as defined in claim 1, the compound of formula (II) as defined inclaim 4, or the cross-linked self-assembled star polymer of formula(III) as defined in claim
 8. 20. A process for the synthesis of thecompound of formula (I) as defined in claim 1, the process comprising:(1) reacting an amine or TFA/BF4 salt initiator of formula (IV) below

wherein I₁ to I₃, R₁, R_(1′) and R_(1″) are as defined in any of theclaims 1-3, with an appropriate N-carboxyanhydride (NCA); alternatively,reacting the amine or tetrafluoroborate or trifluoroacetate ammoniumsalt form of initiator of step (1) with the appropriateN-carboxyanhydrides in a sequential manner to obtain a block co-polymer;alternatively, reacting the amine or tetrafluoroborate ortrifluoroacetate ammonium salt form of initiator of step (1) with anappropriate NCA in a statistical manner to obtain random co-polymers;(2) optionally, reacting the amine group at the N-terminal position withan amine reactive group to introduce R₅, R_(5′) and/or R_(5″); (3)optionally, orthogonally removing amino acid side chains; (4) purifyingthe product obtained in step (1), (2) or (3), optionally byfractionation, precipitation, ultrafiltration, dialysis, size exclusionchromatography or tangential flow filtration.
 21. A process as definedin claim 20, for the synthesis of the compound of formula (II) asdefined in claim 4, the process further comprising: (5) introducing theCL groups at reactive amino acid side chain, at the appropriate molarratio; (6) purifying the product obtained in step (5) optionally byfractionation, precipitation, ultrafiltration, dialysis, size exclusionchromatography or tangential flow filtration.
 22. A process as definedin claim 21, for the synthesis of the cross-linked self-assembled starpolymer of formula (III) as defined in claim 8, the process furthercomprising: (7) reacting the CL groups of the self-assembled compoundsof formula (II) forming nanometric assemblies, to covalently cross-linkthe self-assembled star polymers; (8) purifying the product obtained instep (7) optionally by fractionation, precipitation, ultrafiltration,dialysis, size exclusion chromatography or tangential flow filtration.